Compositions of and methods for in vitro viral genome engineering

ABSTRACT

The present disclosure relates to a method of in vitro engineering of nucleic acids. This disclosure further relates to in vitro engineering of viral genomes and to the improvement of viral properties by in vitro genomic engineering of viral genomes. Specifically, the disclosure relates to in vitro viral genomic digestion using RNA-guided Cas9, the assembly of a recombinant genome by the insertion of a DNA or RNA fragment into the digested viral genome and transformation of a host cell with the recombinant genome. This method also related to in vitro engineering for error correction of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 16/246,381 filed Jan. 11, 2019, which is acontinuation of U.S. Non-Provisional patent application Ser. No.14/970,458 filed Dec. 15, 2015 and issued as U.S. Pat. No. 10,221,398,which is a continuation of International Patent Application No.PCT/US2015/065891 filed Dec. 15, 2015, which claims priority to U.S.Provisional Patent Application No. 62/092,707 filed Dec. 16, 2014, toU.S. Provisional Patent Application No. 62/102,362 filed Jan. 12, 2015,and to U.S. Provisional Patent Application No. 62/242,811 filed Oct. 16,2015, the contents of all of which are hereby incorporated by referencein their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“054249-514CO3US_SL_ST25.txt” created on Jul. 10, 2019 and having a sizeof 141,837 bytes. The contents of the text file are incorporated byreference herein in their entirety.

FIELD OF THE DISCLOSURE

The disclosure is directed generally to the rapid engineering of genomesand more specifically to engineering viral genomes in vitro.

BACKGROUND INFORMATION

Viruses are used in many scientific applications, especially in thedevelopment of prophylactics, therapeutics, and diagnostics. For thesepurposes, viruses are often subjected to genetic engineering. In vivoengineering requires a tractable host organism and can often take weeksto months to create modified viruses and viral vectors (Levin and Bull,Nat Rev Microbiol., 2004 February; 2(2):166-73, incorporated herein byreference). Additionally, there are toxicity concerns inherentlyassociated with the manipulation of many viral genomes in cells. Effortsto develop methods for in vitro genetic engineering of large viralgenomes have thus far been constrained by the availability of uniquerestriction enzyme target sequences and the low efficiencies obtainedfor genome digestion and subsequent recombinant assembly. Furthermore,many genetic engineering efforts are thwarted by incorrectly predictedviral genomic termini. For example, publicly available PB1-like viralgenomes incorrectly place the end sequences in the middle of the genome,an often occurring error using current sequencing and in silico genomeassembly methods (Ceyssens et al., Environ Mibrobiol. 2009 November;11(11):2874-83).

There remains a need for the rapid genetic engineering of viral genomes,especially for viruses infecting non-genetically tractable hosts. Thepresent disclosure utilizes in vitro Cas9 mediated digestion andassembly to site specifically engineer whole viral genomes. This methoddrastically increases the precision, simplicity and speed at which viralgenomes can be genetically modified. Further, this technique overcomesthe well-established difficulty of manipulating often toxic virulentviral genomes inside native and heterologous host cells. Utilizing thedisclosed in vitro engineering method also enables identification ofcorrect viral genomic ends, which facilitates subsequent engineering viathe present disclosure.

In vitro error correction is an invaluable technique for generatingdesired sequences following cloning or assembly techniques. Standarderror correction methods are PCR-based, which has two inherentproblems: 1) PCR can introduce additional unwanted mutations into thenucleic acid and 2) PCR, in this context, has a size restriction ofapproximated 5 kb before it becomes increasingly error prone (QuickChange site-directed mutagenesis kit manual, New England Biolabs, USA).Therefore, standard PCR-based error correction methods cannot reliablybe performed on plasmids larger than 5 kb, either as a result ofadditional PCR-generated mutations or a failure to amplify the completetemplate.

SUMMARY OF THE DISCLOSURE

Among the various aspects of the present disclosure are compositions andmethods for engineering nucleic acid sequences in vitro using anRNA-guided nuclease. In one aspect, the disclosure relates to theimprovement of specific viral properties by in vitro genetic engineeringof viral nucleic acid sequences and the improved viral compositions orparticles. In another aspect, the disclosure relates to the in vitrodigestion of viral nucleic acid sequences using an RNA-guidedendonuclease, e.g., Cas9, followed by the assembly of a recombinantnucleic acid sequence by the insertion of a DNA or RNA fragment(s) intothe digested viral nucleic acid.

In some embodiments, the present disclosure provides an engineered viruscomprising an engineered viral nucleic acid capable, upon introductioninto a host cell, of producing non-naturally occurring viral particleswith two or more improved viral properties compared to the viralparticles produced by introduction of the non-engineered viral nucleicacid into a host cell.

In some aspects, the produced viral particles have at least threeimproved viral properties.

In some aspects, each improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing.

In some aspects, the engineered viral nucleic acid is an engineeredviral genome.

In some aspects, the engineered viral genome is an engineeredbacteriophage genome. In some aspects, at least one of the improvedviral properties is host range.

In some aspects, each improved viral property is the result of at leastone modification in the engineered viral nucleic acid.

In some aspects, at least one improved viral property is the result ofat least two modifications in the engineered viral nucleic acid.

In some aspects, the at least one modification in the engineered viralnucleic acid are the result of a single engineering step.

In some aspects, the at least one modification in the engineered viralnucleic acid are the result of iterative engineering steps.

In some aspects, at least one of the modifications is within a nucleicacid sequence having at least 85% identity to SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:50, or SEQ ID NO:25.

In some aspects, at least one of the modifications is within a nucleicacid sequence encoding an amino acid sequence having at least 85%identity to SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:5, SEQID NO:48, or SEQ ID NO:49.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises all or aportion of a heterologous gp18 gene. In some aspects, the heterologousgp18 gene has at least 85% identity to SEQ ID NO:26. In some aspects,the heterologous gp18 gene encodes an amino acid sequence with at least85% identity to SEQ ID NO:38.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises all or aportion of an engineered gp34 gene. In some aspects, the engineered gp34gene encodes an amino acid sequence comprising a mutation at a positioncorresponding to amino acid position 55 of SEQ ID NO:5.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises amodification in one or more sequences having at least 85% identity to asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, and SEQ ID NO:50. In some aspects, the engineered viralgenome further comprises a modification in each of a sequence having atleast 85% identity to SEQ ID NO:1, a sequence having at least 85%identity to SEQ ID NO:2, a sequence having at least 85% identity to SEQID NO:3, and a sequence having at least 85% identity to SEQ ID NO:50. Insome aspects, the modifications comprise a G to A replacement at aposition corresponding to nucleic acid position 50 of SEQ ID NO:1, a Gto T replacement at a position corresponding to nucleic acid position160 of SEQ ID NO:50, a A to G replacement at a position corresponding tonucleic acid position 245 of SEQ ID NO:2, a AT to TC replacement atpositions corresponding to nucleic acid positions 247-248 of SEQ IDNO:2, and a A to G replacement at a position corresponding to nucleicacid position 757 of SEQ ID NO:3.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises amodification in one or more nucleic acid sequences encoding an aminoacid sequence having at least 85% identity to a sequence selected fromthe group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, andSEQ ID NO:48. In some aspects, the engineered viral genome comprises amodification in a nucleic acid sequence encoding each of an amino acidsequence having at least 85% identity to SEQ ID NO:34, an amino acidsequence having at least 85% identity to SEQ ID NO:35, an amino acidsequence having at least 85% identity to SEQ ID NO:36, and an amino acidsequence having at least 85% identity to SEQ ID NO:48. In some aspects,the modifications comprise a C to Y replacement at a positioncorresponding to amino acid position 17 of SEQ ID NO:34, a D to Yreplacement at a position corresponding to amino acid position 36 of SEQID NO:48, a D to G replacement at a position corresponding to amino acidposition 82 of SEQ ID NO:35, a I to S replacement at positioncorresponding to amino acid position 83 of SEQ ID NO:35, and a N to Dreplacement at a position corresponding to amino acid position 253 ofSEQ ID NO:36.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises amodification within a sequence having at least 85% identity to SEQ IDNO:25. In some aspects, the modification is an insertion of aheterologous nucleic acid molecule into a sequence having at least 85%identity to SEQ ID NO:25, or a replacement of a sequence comprisedwithin a sequence having at least 85% identity to SEQ ID NO:25 with aheterologous nucleic acid molecule. In some aspects, the heterologousnucleic acid molecule comprises a heterologous nucleic acid sequencehaving at least 85% identity to a sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 85% identity to the LUZ19 genome. Insome aspects, the engineered viral genome further comprises amodification within a nucleic acid sequence encoding an amino acidsequence having at least 85% identity to SEQ ID NO:49. In some aspects,the modification is an insertion of a heterologous nucleic acid moleculeinto a nucleic acid sequence encoding an amino acid sequence having atleast 85% identity to SEQ ID NO:49, or a replacement of a nucleic acidsequence comprised within a nucleic acid sequence encoding an amino acidsequence having at least 85% identity to SEQ ID NO:49 with aheterologous nucleic acid molecule. In some aspects, the heterologousnucleic acid molecule comprises a heterologous nucleic acid sequenceencoding an amino acid sequence having at least 85% identity to asequence selected from the group consisting of SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, and SEQ ID NO:47.

In some aspects, the engineered viral nucleic acid comprises aheterologous nucleic acid sequence operably linked to a promotercomprising a nucleic acid sequence comprised within SEQ ID NO:21 or aportion thereof.

In some aspects, the engineered viral nucleic acid comprises aheterologous nucleic acid sequence operably linked to a terminatorcomprising the nucleic acid sequence of SEQ ID NO:22 or a portionthereof.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties comprising: (a) providing a first viral genome; and (b)generating an engineered viral genome by combining at least one fragmentof the first viral genome with at least one repair nucleic acid moleculeto generate a second viral genome comprising at least one modificationcompared to the first viral genome; wherein, the second viral genome,upon being introduced into a host cell, is capable of producing viralparticles with two or more improved viral properties.

In some aspects, the method further comprises (c) repeating steps(a)-(b) in one or more iterations.

In some aspects, each improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing.

In some aspects, improved property or improved properties and improvedviral property or improved viral properties are used interchangeably.

In some aspects, generating the engineered viral genome in step (b)comprises: (1) in vitro digestion of a region of the first viral genomeusing an endonuclease; and (2) assembling at least one fragment of thedigested first viral genome with at least one repair nucleic acidmolecule.

In some aspects, the first viral genome is isolated from viralparticles.

In some aspects, the first viral genome or the at least one repairnucleic acid molecule is synthesized de novo.

In some aspects, de novo synthesis comprises combining chemicallysynthesized nucleic acid molecules, PCR-amplified nucleic acidsequences, digested fragments of isolated nucleic acid molecules, or anycombination thereof.

In some aspects, the first viral genome or the at least one repairnucleic acid molecule is amplified prior to in vitro digestion.

In some aspects, the first viral genome at least 3 kb, at least 10 kb,at least 18 kb, at least 25 kb, or at least 30 kb.

In some aspects, the assembly is performed in vitro or in vivo.

In some aspects, the assembly is performed in vitro with a mixturecomprising: (a) an isolated 5′ to 3′ exonuclease that lacks 3′exonuclease activity; (b) an isolated non-strand-displacing DNApolymerase with 3′ exonuclease activity, or a mixture of said DNApolymerase with a second DNA polymerase that lacks 3′ exonucleaseactivity; (c) an isolated ligase; and (d) a mixture of dNTPs, underconditions that are effective for insertion of the fragment into thedigested viral nucleic acid to form a recombinant nucleic acidcomprising the engineered viral genome.

In some aspects, the endonuclease is an RNA-guided nuclease.

In some aspects, the method further comprises at least one guiding RNA.

In some aspects, the RNA-guided nuclease is Cas9 or a Cas9 derivedenzyme and wherein the at least one guiding RNA comprises 1) a chimericgRNA or 2) a crRNA and tracrRNA.

In some aspects, the endonuclease is heat inactivated or removed priorto assembly.

In some aspects, the in vitro digestion further comprises spermidine.

In some aspects, the method further comprises transforming theengineered viral genome into a host cell.

In some aspects, the method further comprises using an in vitropackaging kit for packaging of the engineered viral genome into viralparticles.

In some embodiments, the present disclosure provides an engineered virusgenerated by any of the methods disclosed herein. In some aspects, theengineered virus is any of the engineered viruses disclosed herein.

In some embodiments, the present disclosure provides a kit forengineering viral nucleic acid molecules comprising: (a) purifiedrecombinant RNA-guided nuclease; (b) an isolated 5′ to 3′ exonucleasethat lacks 3′ exonuclease activity; (c) an isolatednon-strand-displacing DNA polymerase with 3′ exonuclease activity, or amixture of said DNA polymerase with a second DNA polymerase that lacks3′ exonuclease activity; and (d) an isolated thermostable ligase.

In some aspects, the kit further comprises one or more of: (1) acrowding agent; (2) a mixture of dNTPs; and (3) a suitable buffer.

In some aspects, the kit further comprises custom-designed guide RNAs.

In some aspects, the kit further comprises custom-designed synthesizednucleic acid molecules to serve as the inserted DNA fragment in anassembly reaction.

In some aspects, the kit further comprises competent host cells fortransformation.

In some aspects, the kit further comprises isolated viral genomicnucleic acids.

In some embodiments, the present disclosure provides an in vitroengineered viral nucleic acid system comprising: isolated viral nucleicacid, recombinant RNA-guided nuclease, at least one guiding RNA, and anucleic acid fragment to be inserted into the isolated nucleic aciddigestion site.

In some aspects, the system is such that the recombinant RNA-guidednuclease and at least one targeting RNA form a complex capable ofdigesting the isolated viral nucleic acid.

In some aspects, the system further comprises spermidine.

In some aspects, the system further comprises: an isolated 5′ to 3′exonuclease that lacks 3′ exonuclease activity; an isolatednon-strand-displacing DNA polymerase with 3′ exonuclease activity, or amixture of said DNA polymerase with a second DNA polymerase that lacks3′ exonuclease activity; an isolated ligase; and a mixture of dNTPs,wherein the system is under conditions that are effective for insertionof the nucleic acid fragment into the isolated viral nucleic acid at thesite of RNA-guided nuclease digestion to form a recombinant viralnucleic acid.

In some aspects, the herein described system is such that therecombinant viral nucleic acid is capable of producing non-naturallyoccurring viral particles with at least two improved viral propertiescompared to viral particles resulting from the non-engineered viralnucleic acid. In some examples, the improved viral property orproperties are selected from the group consisting of host range, virallytic cycle, adsorption, attachment, injection, replication andassembly, lysis, burst size, immune evasion, immune stimulation, immunedeactivation, biofilm dispersion, bacterial phage resistance, bacterialantibiotic sensitization, modulation of virulence factors, and targetedhost genome digestion or editing.

In some aspects, in the herein described system, the RNA-guided nucleaseis Cas9 or a Cas9-derived enzyme. In some aspects, the RNAguided-nuclease is inactivated or removed prior to assembly.

In some embodiments, the present disclosure provides a method ofengineering a nucleic acid sequence comprising: (a) providing a nucleicacid; (b) in vitro digestion of a region of the nucleic acid using anRNA-guided nuclease; and (c) assembly of a recombinant nucleic acid bythe insertion of a DNA fragment into the digested nucleic acid, whereinthe assembly is performed in vitro in a single vessel with a mixture ofcomponents comprising: (i) an isolated 5′ to 3′ exonuclease that lacks3′ exonuclease activity; (ii) an isolated non-strand-displacing DNApolymerase with 3′ exonuclease activity, or a mixture of said DNApolymerase with a second DNA polymerase that lacks 3′ exonucleaseactivity; (iii) an isolated ligase; and (iv) a mixture of dNTPs, underconditions that are effective for insertion of the fragment into thedigested nucleic acid to form a recombinant nucleic acid.

In some aspects, the RNA-guided nuclease is Cas9 or a Cas9 derivedenzyme. In some examples, the RNA-guided nuclease is inactivated byexposure to heat or removed prior to assembly.

In some aspects, the method further comprises: (d) transformation of therecombinant nucleic acid into a host cell.

In some aspects, the present disclosure provides a method of engineeringa nucleic acid wherein the nucleic acid is a plasmid isolated from ahost cell. In some aspects, the plasmid is at least 5 kb. In someaspects, the plasmid is at least 6 kb. In some aspects, the plasmid isat least 10 kb. In some aspects, the plasmid is at least 15 kb. In someaspects, the plasmid is at least 20 kb.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F shows a schematic of the in vitro process to directly engineerviral genomes. A) Genomes are extracted from purified viral particles,utilizing methods known to those skilled in the art. Grey linesillustrate an example dsDNA viral genome. Light grey lines at the genometermini denote direct terminal repeats commonly found in many viralgenomes. B) Viral genomes are then digested at one or more locationssite specifically using an RNA-guided nuclease, such as Cas9, coupledwith purified targeting RNAs such as chimeric gRNAs, crRNAs andtracrRNAs, or crRNAs alone. Illustration depicts RNA-guided nucleasetargeting defined viral genomic locations, as specified by the givenRNAs. C) The RNA-guided nuclease is inactivated using methods known inthe art including but not limited to, exposure to heat and/or removedusing classic phenol-chloroform extraction. D) A DNA or RNA insert isobtained using methods known in the art including but not limited to, invitro synthesis, amplification (PCR), or enzyme mediated liberation fromplasmids, viruses, or bacterial genomic DNA (gDNA). Diagram depictsnewly generated insert (dark grey lines) with homology regionscorresponding to viral sequences flanking the RNA-guided nucleasedigestion site(s) (grey terminal regions). E) Digested viral genomes andpurified insert are assembled in vitro using methods known in the artincluding but not limited to, Gibson Assembly, SLIC, and/or Golden GateAssembly. Illustration depicting the assembled recombinant genome, nowharboring the new insert sequence (dark grey lines) at the desiredlocation. F) Recombinant viral genomes are transformed directly intohost cells using methods known in the art including but not limited to,electroporation or chemical transformation. Cartoon shows the recoveryof functional viral particles following transformation of an infectiveviral genome into susceptible host cells.

FIG. 2A-F show the in vitro engineering of a viral genome. A)Purification of ˜43 kb dsDNA LUZ19 viral genome directly from viralparticles. B) Site-specific digestion of purified viral genome at twoindependent locations to remove gp7 gene fragment using RNA-dependentnuclease Cas9 and in vitro transcribed gRNAs. C) PCR was used to amplifygp7 gene from the virus ΦKF77. D) In vitro Gibson Assembly was used tosequence specifically integrate the PCR amplified 1KF77 gp7 genefragment seamlessly into the digested LUZ19 genome. E) Infectious invitro assembled genomes were transformed directly into host cells torecover functional viral particles, evidenced by plaque formation. F)Internal and external primers were used to PCR verify that virusescontained the new DNA fragment at the correct genomic site. All testedviral clones were PCR positive for the new insert ΦKF77 gp7 fragment(right 7 lanes).

FIG. 3A-B shows the generation of a virus with improved viral propertiesfollowing in vitro viral genome engineering. A) Diagram depicting thegenomes of the natural LUZ19 virus and an engineered derivativeharboring the LKD16 virus gp18 gene in place of the natural LUZ19 gp18sequence. Black arrows denote the native LUZ19 open reading frames,while the grey arrow indicates the newly integrated LKD16 gp18 gene. B)Left, Venn diagram showing the shared and independent host bacteriainfected by LUZ19 and LKD16 viruses. A diverse collection of 282 P.aeruginosa clinical isolates were tested. Right, Venn diagram showingthat an engineered LUZ19 virus harboring the LKD16 gp18 gene has anexpanded host range, including 3 of the 6 strains previously onlyinfected by LKD16.

FIG. 4A-C are schematics showing the process used to identify and selectthe genetic elements and point mutations required for host rangeexpansion and engineering a wide host range virus capable of infectingthe full host range of a viral genus. A) Schematic representation of theprocess used to identify mutations responsible for host rangespecificity. B) Schematic representation depicting the genomemodifications required to generate a wide host range LUZ19 (WHR LUZ19)virus; asterisks (*) identify the location of each point mutationrelated to host range. Labels gp13 C17Y, gp18 D36Y, gp38 D82G and I83S,and gp40 N253D describe the gene products and amino acid point mutationlinked to LUZ19 host range expansion. PA7245, PA7255, PA7410, PA7427,PA7503 and PA7686 are P. aeruginosa clinical isolates susceptible onlyto LKD16 and WHR LUZ19; PA7649 is a P. aeruginosa clinical isolatesensitive only to ΦKMV and WHR LUZ19. Clinical isolates infectedfollowing addition of a given mutation are depicted above the givenmutation. C) Left, Venn diagram showing the shared and independent hostbacteria infected by LUZ19, LKD16, and ΦKMV viruses. Right, Venn diagramshowing that the engineered WHR LUZ19 virus harboring the pointmutations described above is able to infect all 67 strains susceptibleto the ΦKMV genus of viruses.

FIG. 5A-E shows that mutation of LUZ19 Gp34 protein improves lyticactivity. A) The LUZ19 Gp34 protein is a member of the viral tailtubular complex (see inlaid image). B) Soft agar plaque assay for tworelated phage expressing either the wild type LUZ19 Gp34 or Gp34 deltaLeucine 55 (L55Δ) mutation (Phage*). Images were taken over a two-dayperiod, and illustrate that phage expressing a Gp34 L55Δ mutation haveincreased zones of lysis. C) Crystal violet biofilm assay extrapolatingbiofilm biomass as a measure of the incorporation of crystal violet. TheLUZ19* phage expressing Gp34 L55Δ was better able to disrupt P.aeruginosa biofilm preformed for 8 hours as compared to wild type LUZ19.Gentamicin at tenfold the minimum inhibitory concentration (MIC) wasused to completely remove biofilm. D) Illustration showing the locationof the gp34 mutation as compared to the wild type LUZ19 genome. E) Tabledemonstrating difference in absorption and burst size between LUZ19 andLUZ19 expressing Gp34 L55Δ.

FIG. 6A-F are schematics showing iterative engineering of a virus withimprovement to two independent properties. A) Schematic representationof LUZ19_(LKD16gp18) viral gDNA in which the wild type LUZ19 gp18 genewas replaced with the LKD16 homolog. In black, wild type LUZ19 genomicsequence; in grey, gp18 from LKD16. B) Susceptibility of laboratory andMDR clinical isolates to purified parental (LKD16 and LUZ19) andLUZ19_(LKD16gp18) engineered virus, demonstrating consolidation of hostrange. C) Schematic representation of LUZ19*_(LKD16gp18) viral gDNA inwhich both the leucine encoded at position 55 of Gp34 was deleted andLUZ19 gp18 was replaced with gp18 from virus LKD16. In black, wild typeLUZ19 genomic sequence; in grey, gp18 from LKD16; grey star denotesgp34_(ΔLeu55). D) Susceptibility of laboratory and MDR clinical isolatesto purified parental (LKD16, LUZ19 and LUZ19_(LKD16gp18) harboring gp18from virus LKD16) and engineered virus (LUZ19* harboring a deletion ofthe leucine encoded at position 55 of GP34 and LUZ19*_(LKD16gp18)),demonstrating consolidation of host range in LUZ19_(LKD16gp18) andLUZ19*_(LKD16gp18) viruses. E) Evaluation of wild type and engineeredphage lytic activity against bacteria attached to keratinocytemonolayers. The number of PA01K and PA7245 bacteria attached to cellswas reported as a percentage of the total bacteria incubated withkeratinocyte monolayers. Data demonstrates improved lytic activity ofLUZ19* and LUZ19*_(LKD16gp18) viruses. F) Improved PAO1K and PA72458-hour early biofilm disruption by engineered phage compared to parentalviruses. Gentamicin at tenfold the minimum inhibitory concentration wasused to completely remove biofilm. The data shown are representative ofthree individual experiments performed in triplicate. Bars representmean±SEM; *P<0.01; **P<0.001; ***P<0.0001.

FIG. 7A-F are schematics showing a second example of iterativelyengineering a virus with improvement to two independent properties. A)Schematic representation of LUZ19 engineered to express variousgenetically encoded payloads from an improved gp49 locus. The gp49 genewas replaced by a cassette containing a gene of interest (GOI) flankedby the major capsid (gp32) promoter and terminator (P_(gp32) andT_(gp32)). Biofilm dispersing GOI utilized: EPS depolymerases (Ppl5gp44tail spike gp44 from Pseudomonas pudita φ15; NTUgp34 tail spike gp34from Klebsiella pneumoniae phage NTUH-K2044-K1-1 (NTU); LKA1gp49-tailspike gp49 from P. aeruginosa phage LKA1), surfactant phenol solublemodulins from Staphylococcus epidermidis (PSMa) and Staphylococcusaureus (PSMa3 and PSMb2), and DspB surfactin from Aggregatibacteractinomycetemcomitans. B) Biofilm dispersion assay showing engineeredLUZ19 phage activity against a 24 h P. aeruginosa PAO1K biofilm treatedwith 100 phage for 3 h. Gentamicin was used at tenfold the minimuminhibitory concentration (MIC). C) Schematic representation of thepreviously engineered WHR LUZ19 phage further engineered to express aGOI from the modified gp49 locus. D) Biofilm dispersion assay showingengineered WHR LUZ19 further modified to express enzymes and surfactinswith activity against a 24 h P. aeruginosa PAO1K biofilm treated with100 phage for 3 h. Engineered payloads: EPS depolymerase Pp15gp44 andSePSMa. Gentamicin was used at tenfold the MIC. E) Susceptibility oflaboratory and clinical isolates to purified parental (LKD16 and LUZ19)and LUZ19 derivatives demonstrating consolidation and maintenance ofhost range upon further engineering to express biofilm-dispersingmoieties. F) Venn diagram showing the retention of WHR LUZ19 host rangeafter addition of biofilm dispersing payloads Pp15gp44 and SePSMa.

FIG. 8A-C are schematics showing the creation of a virus able to impedehost cells from acquiring viral resistance when combined withsub-inhibitory concentration of antibiotic. A) Schematic representationof wild type LUZ19 engineered to express the lysins from either MS2 orPRR1 phage. B) and C) Time-kill assays demonstrating sensitization of P.aeruginosa PAO1K to sub-inhibitory concentrations of carbenicillin(Cb−⅕×MIC) by LUZ19 expressing lysins from ssRNA bacteriophage. Thesedata demonstrate that an engineered bacteriophage expressing non-nativelysins in combination with sub-inhibitory antibiotic concentrations canprevent bacteria from rapidly acquiring resistance to a single virus.

FIG. 9A-D are schematics showing the creation of a 2^(nd) virus able toimpede host cells from acquiring viral resistance. A) Schematicrepresentation of wild type LUZ19 engineered to express the bacteriocinprotein PyoS5 from the modified gp49locus. B) Time-kill assays showingthat the growth of XDRPA strain PA7416 was initially inhibited by wildtype LUZ19, however, bacteria rapidly evade the virus leading tobacterial regrowth. Approximately 1×10⁷ cfu were added per well. HighMOI=10 pfu/cfu and low MOI=0.01 pfu/cfu of indicated virus or vehiclewere added at time 0 h. C) Time-kill assays showing that LUZ19 encodingPyoS5 is able to inhibit XDRPA strain PA7416 growth and re-growthrelative to the wild type virus. Approximately 1×10⁷ cfu were added perwell. High MOI=10 pfu/cfu and low MOI=0.01 pfu/cfu of indicated virus orvehicle were added at time 0 h. D) Comparison of PA7416 growth after 24hours in the presence of either wild type LUZ19 or LUZ19+pyoS5. Graphdepicts data for low MOI experiment.

FIG. 10 is a schematic drawing of a system integrating targeted viralgenome editing with bacteriophage phenotypic screening to create agenetically modified bacteriophage with improvements in two or morecharacteristics. The system relies on iterative rounds of screening andsequencing mutant or natural viruses with desirable phenotypic traitsand integrating those traits into one or more viral chassis in a singleor multiple engineering steps. This process provides a direct andrational method of rapidly identifying the genetic elements underlying aspecific bacteriophage phenotypic trait, integrating multipleindependent mutations or alleles into a single bacteriophage genome, andcreation of an engineered virus combining two or more improved traits.

FIG. 11A-G shows in vitro engineering of E. coli phage M13 genome. A)Schematic representation of E. coli temperate phage M13mp18 andM13_(paprika). B) Gel electrophoresis of in vitro digested circular M13genomic DNA using gRNAs 1 and 2 in independent reactions with theRNA-guided endonuclease Cas9. Diagram below gel depicts the circular andlinear nature undigested and double digested M13 genomes, respectively.These data demonstrate that both gRNAs accurately and completely digestthe M13 dsDNA at the correct locations. C) Gel electrophoresis of invitro digested circular M13 genomic DNA using both gRNAs and theRNA-guided endonuclease Cas9 in the same reaction (double digest).Diagram below gel depicts the circular and linear nature of undigestedand double digested M13 genomes, respectively. D) Gel electrophoresisshowing PCR generated insert containing paprika fluorescent reporter. E)Transformation of in vitro digested and assembled engineeredM13_(paprika) gDNA into E. coli cells to recover functional viralparticles. Viral plaques are feint and veiled because M13 is a temperatebacteriophage that does not lyse host cells, resulting in poor plaqueformation. Undigested M13 gDNA was used as a positive control. M13 gDNAdigested with Cas9, but assembled in the absence of an insert (noinsert) demonstrates the both the completeness of the digestion and thelow level of background. F) Plaque PCR verification of M13_(paprika)engineering. Forward and reverse primers were designed external toinsert homology regions. Non-engineered M13 gDNA produced a 0.9 kbproduct and was used as a negative control for PCR reactions. G)Fluorescent (bottom) and bright filed (top) images of parental andengineered M13_(paprika) paprika during plaque formation.

FIG. 12A-E shows in vitro engineering of a second E. coli phage genome.A) Schematic representation of E. coli phage λ ΔcII. The linear phagegenome is 48.5 kb in size. B) Gel electrophoresis of in vitro digested λgenomic DNA using gRNAs 1 and 2 in independent reactions with anRNA-guided endonuclease. Diagram below gel depicts the linear undigestedand expected digestion products. These data demonstrate that both gRNAsaccurately and completely digest the λ dsDNA at the correct locations.C) Gel electrophoresis of in vitro double digested λ genomic DNA usingboth gRNAs and an RNA-guided endonuclease in the same reaction. Diagrambelow gel depicts the linear undigested and expected double digestionproducts. D) Schematic depicting the use of phage λ packaging buffer topackage wild type and recombinant phage genomes in vitro. Cas9 doubledigested and assembled phage λ genomes were in vitro packaged accordingto manufacturer's protocol and plated on E. coli to recover newlyengineered λ ΔcII phage. E) PCR verification of λ ΔcII gene. Forwardprimer was located external to the region of engineering. Deletionpositive clones have an expected size of 300 bp.

FIG. 13A-D shows in vitro engineering of sequences from a humancytomegalovirus virus (HCMV). A) Schematic representation of 235 kb fulllength HCMV viral genome. Top cigar shaped genome represents full lengthgenome, while black section denotes region of manipulation. Small whitesection denotes 235 bp insertion being added using the herein describedin vitro engineering method. B) Gel electrophoresis of in vitro doubledigested plasmid harboring 17.8 kb region of HCMV genome using two gRNAsand RNA-guided endonuclease Cas9. Diagram below gel depicts the circularundigested and linear double digestion products. These data demonstratethat both gRNAs accurately and completely digest the HCMV dsDNA sequenceat the correct location. C) Gel electrophoresis showing PCR generatedinsert containing new RL13 insertion sequence. D) PCR verification ofmodified HCMV sequence. Forward primer was located external to theregion of engineering. Insertion positive clones have an expected sizeof 500 bp.

FIG. 14A-F shows rapid identification of phage ends. A) Isolation ofgenomic DNA from purified viral particles. B) Next-generation sequencingof gDNA (MiSeq or PacBio) and automated merging of high quality DNAreads into longer assemblies to reconstruct the original sequence. Inlight grey, the DTRs—direct terminal repeats. Automated assemblysoftware incorrectly places the DTRs of terminally repetitive genomes inthe internal region of viral sequence. Genomic physical ends areconfirmed by targeted Cas9 digestion of the predicted sequence. C) Insilico prediction of physical genome ends based on identification ofdouble coverage sequencing regions and BLAST search that matches aclosely related terminally repeated genome. Physical ends are confirmedby Cas9 endonuclease cleavage of predicted physical ends. D) After Cas9inactivation, DNA fragments corresponding to the genomic physical endsare purified and sequenced. E) Accurate genome assembly based onphysical ends sequencing. F) Example of genomic physical ends mapping ofLBL3 and 14-1 phage (terminally repetitive genomes) using Cas9 targeteddigestion at specific position predicted by in silico genomerearrangements. Light grey arrows point to the DNA fragments purifiedand sequenced.

FIG. 15A-C schematic drawing of chimeric sgRNA design and synthesisstrategy. A) Illustration showing the location of NGG PAM motifs (darkgrey underlined sequences) and sgRNA target sites (light grey sequences)flanking a gene of interest (GOI). Black sequences denote remainingviral genomic sequences. B) Design of oligonucleotides used as templatesfor in vitro transcription of sgRNAs. Sequences constituting the T7promoter, sgRNA targeting sequence, and conserved chimeric sgRNA regionare denoted in underlined dark grey, light grey, and black text,respectively. C) Diagram of in vitro transcribed chimeric sgRNA. Lightgrey and black sequences indicate the targeting and conserved chimericregions constituting each functional sgRNA, respectively. All Ns denotevariable sequences used to alter the target specificity of each sgRNA.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure provides compositions of and methods for in vitroengineering and further relates to the improvement of viral properties.The present disclosure further provides a method for in vitroengineering of nucleic acids.

Before the present compositions and methods are described, it is to beunderstood that this disclosure is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyin the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although, any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the disclosure, the preferred methods andmaterials are now described. The definitions set forth below are forunderstanding of the disclosure but shall in no way be considered tosupplant the understanding of the terms held by those of ordinary skillin the art.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

As used herein, the terms “about” or “approximately” when referring toany numerical value are intended to mean a value of plus or minus 10% ofthe stated value. For example, “about 50 degrees C.” (or “approximately50 degrees C.”) encompasses a range of temperatures from 45 degrees C.to 55 degrees C., inclusive. Similarly, “about 100 mM” (or“approximately 100 mM”) encompasses a range of concentrations from 90 mMto 110 mM, inclusive. Alternatively, “about” or “approximately” can meanwithin 5% of the stated value, or in some cases within 2.5% of thestated value, or, “about” can mean rounded to the nearest significantdigit. All ranges provided within the application are inclusive of thevalues of the upper and lower ends of the range.

The terms, “cells”, “cell cultures”, “cell line”, “recombinant hostcells”, “recipient cells” and “host cells” as used herein, include theprimary subject cells and any progeny thereof, without regard to thenumber of transfers. It should be understood that not all progeny areexactly identical to the parental cell (due to deliberate or inadvertentmutations or differences in environment); however, such altered progenyare included in these terms, so long as the progeny retain the samefunctionality as that of the originally transformed cell.

The term “assembly” or “assemble” as used herein refers to the joiningof DNA or RNA molecules.

The term “repair nucleic acid molecule” as used herein refers to anucleic acid molecule capable of being assembled with one or more DNAfragments or a digested or cleaved DNA plasmid or DNA nucleic acidmolecule in order to generate a contiguous nucleic acid sequencemolecule or closed plasmid DNA.

The terms “de novo synthesis”, “de novo assembly”, “chemical synthesis”,and “DNA synthesis” refer to methods of creating nucleic acid sequenceswithout the need for a pre-existing precursor template.

In those methods of the invention that are carried out “in vitro”, allof the protein components are isolated and/or substantially purified.The in vitro assembly reactions are not carried out in a living cell orwith a crude cell extract; the reactions are carried out in a cell-freeenvironment.

A “functional RNA molecule” is an RNA molecule that can interact withone or more proteins or nucleic acid molecules to perform or participatein a structural, catalytic, or regulatory function that affects theexpression or activity of a gene or gene product other than the genethat produced the functional RNA. A functional RNA can be, for example,a transfer RNA (tRNA), ribosomal RNA (rRNA), anti-sense RNA (asRNA),microRNA (miRNA), short-hairpin RNA (shRNA), small interfering RNA(siRNA), a guide RNA (gRNA), crispr RNA (crRNA), or transactivating RNA(tracrRNA) of a CRISPR system, small nucleolar RNAs (snoRNAs),piwi-interacting RNA (piRNA), or a ribozyme.

The term “gene” is used broadly to refer to any segment of a nucleicacid molecule (typically DNA, but optionally RNA) encoding a polypeptideor expressed RNA. Thus, genes include sequences encoding expressed RNA(which can include polypeptide coding sequences or, for example,functional RNAs, such as ribosomal RNAs, tRNAs, antisense RNAs,microRNAs, short hairpin RNAs, gRNAs, crRNAs, tracrRNAs, ribozymes,etc.). Genes may further comprise regulatory sequences required for oraffecting their expression, as well as sequences associated with theprotein or RNA-encoding sequence in its natural state, such as, forexample, intron sequences, 5′ or 3′ untranslated sequences, etc. In someexamples, a gene may only refer to a protein-encoding portion of a DNAor RNA molecule, which may or may not include introns. A gene ispreferably greater than 50 nucleotides in length, more preferablygreater than 100 nucleotide in length, and can be, for example, between50 nucleotides and 500,000 nucleotides in length, such as between 100nucleotides and 100,000 nucleotides in length or between about 200nucleotides and about 50,000 nucleotides in length, or about 200nucleotides and about 20,000 nucleotides in length. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information.

The term “nucleic acid” or “nucleic acid molecule” refers to, a segmentof DNA or RNA (e.g., mRNA), and also includes nucleic acids havingmodified backbones (e.g., peptide nucleic acids, locked nucleic acids)or modified or non-naturally-occurring nucleobases. The nucleic acidmolecules can be double-stranded or single-stranded; a single strandednucleic acid that comprises a gene or a portion thereof can be a coding(sense) strand or a non-coding (antisense) strand.

The terms “coding sequence” or “coding region” as used herein, refer toregions of a nucleic acid sequence which can be transcribed to produce afunctional RNA or an RNA transcript that can be translated into apolypeptide when placed under the control of appropriate expressioncontrol sequences and in the presence of appropriate cellular machineryor enzymes. The term “non-coding sequence” or “non-coding region” refersto regions of a nucleic acid sequence that are not transcribed andtranslated into amino acids (e.g., introns, untranslated regions, etc.)or are not transcribed or do not form at least a portion of a maturefunctional RNA sequence.

As used herein, the term “protein” or “polypeptide” is intended toencompass a singular “polypeptide” as well as plural “polypeptides,” andrefers to a molecule composed of monomers (amino acids) linearly linkedby amide bonds (also known as peptide bonds). The term “polypeptide”refers to any chain or chains of two or more amino acids, and does notrefer to a specific length of the product. Thus, peptides, dipeptides,tripeptides, oligopeptides, “protein,” “amino acid chain,” or any otherterm used to refer to a chain or chains of two or more amino acids, areincluded within the definition of “polypeptide,” and the term“polypeptide” can be used instead of, or interchangeably with any ofthese terms.

A nucleic acid molecule may be “derived from” an indicated source, whichincludes the isolation (in whole or in part) of a nucleic acid segmentfrom an indicated source. A nucleic acid molecule may also be derivedfrom an indicated source by, for example, direct cloning, PCRamplification, or artificial synthesis from the indicated polynucleotidesource or based on a sequence associated with the indicatedpolynucleotide source. Genes or nucleic acid molecules derived from aparticular source or species also include genes or nucleic acidmolecules having sequence modifications with respect to the sourcenucleic acid molecules. For example, a gene or nucleic acid moleculederived from a source (e.g., a particular referenced gene) can includeone or more mutations with respect to the source gene or nucleic acidmolecule that are unintended or that are deliberately introduced, and ifone or more mutations, including substitutions, deletions, orinsertions, are deliberately introduced the sequence alterations can beintroduced by random or targeted mutation of cells or nucleic acids, byamplification or other molecular biology techniques, or by chemicalsynthesis, or any combination thereof. A gene or nucleic acid moleculethat is derived from a referenced gene or nucleic acid molecule thatencodes a functional RNA or polypeptide can encode a functional RNA orpolypeptide having at least 75%, at least 80%, at least 85%, at least90%, or at least 95%, sequence identity with the referenced or sourcefunctional RNA or polypeptide, or to a functional fragment thereof. Forexample, a gene or nucleic acid molecule that is derived from areferenced gene or nucleic acid molecule that encodes a functional RNAor polypeptide can encode a functional RNA or polypeptide having atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity with the referenced orsource functional RNA or polypeptide, or to a functional fragmentthereof.

As used herein, an “isolated” nucleic acid or protein is removed fromits natural milieu or the context in which the nucleic acid or proteinexists in nature. For example, an isolated protein or nucleic acidmolecule is removed from the cell or organism with which it isassociated in its native or natural environment. An isolated nucleicacid or protein can be, in some instances, partially or substantiallypurified, but no particular level of purification is required forisolation. Thus, for example, an isolated nucleic acid molecule can be anucleic acid sequence that has been excised from the chromosome, genome,or episome that it is integrated into in nature.

A “purified” nucleic acid molecule or nucleotide sequence, or protein orpolypeptide sequence, is substantially free of cellular material andcellular components. The purified nucleic acid molecule or protein maybe free of chemicals beyond buffer or solvent, for example.“Substantially free” is not intended to mean that other componentsbeyond the novel nucleic acid molecules are undetectable.

The terms “naturally-occurring” and “wild type” refer to a form found innature. For example, a naturally occurring or wild type nucleic acidmolecule, nucleotide sequence or protein may be present in and isolatedfrom a natural source, and is not intentionally modified by humanmanipulation.

As used herein, “expression” includes the expression of a gene at leastat the level of RNA production, and an “expression product” includes theresultant product, e.g., a polypeptide or functional RNA (e.g., aribosomal RNA, a tRNA, an antisense RNA, a micro RNA, an shRNA, aribozyme, etc.), of an expressed gene. The term “increased expression”includes an alteration in gene expression to facilitate increased mRNAproduction and/or increased polypeptide expression. “Increasedproduction”, when referring to protein abundance or the abundance ofactive protein resulting from gene expression, protein turnover rates,protein activation states, and the like, includes an increase in theamount of polypeptide expression, in the level of the enzymatic activityof a polypeptide, or a combination of both, as compared to the nativeproduction or enzymatic activity of the polypeptide.

“Exogenous nucleic acid molecule” or “exogenous gene” refers to anucleic acid molecule or gene that has been introduced (“transformed”)into a cell or virus. A transformed organism may be referred to as arecombinant cell or virus, into which additional exogenous gene(s) maybe introduced. A descendent of a cell or virus transformed with anucleic acid molecule is also referred to as “transformed” or“recombinant” if it has inherited the exogenous nucleic acid molecule.The exogenous gene may be from a different species (and so“heterologous”), or from the same species (and so “homologous”),relative to the organism being transformed. An “endogenous” nucleic acidmolecule, gene or protein is a native nucleic acid molecule, gene orprotein as it occurs in, or is naturally produced by, the organism.

Further, the term “exogenous” as used herein in the context of a gene orprotein, refers to a gene or protein that is not derived from the hostorganism species.

The term “transgene” as used herein, refers to an exogenous gene, thatis, a gene introduced into a microorganism or a progenitor by humanintervention.

The term “ortholog” of a gene or protein as used herein refers to itsfunctional equivalent in another species.

Gene and protein Accession numbers, commonly provided herein inparenthesis after a gene or species name, are unique identifiers for asequence record publicly available at the National Center forBiotechnology Information (NCBI) web site (ncbi.nlm.nih.gov) maintainedby the United States National Institutes of Health. The “GenInfoIdentifier” (GI) sequence identification number is specific to anucleotide or amino acid sequence. If a sequence changes in any way, anew GI number is assigned. A Sequence Revision History tool is availableto track the various GI numbers, version numbers, and update dates forsequences that appear in a specific GenBank record. Searching andobtaining nucleic acid or gene sequences or protein sequences based onAccession numbers and GI numbers is well known in the arts of, e.g.,cell biology, biochemistry, molecular biology, and molecular genetics.

As used herein, the terms “percent identity” or “homology” with respectto nucleic acid or polypeptide sequences are defined as the percentageof nucleotide or amino acid residues in the candidate sequence that areidentical with the known polypeptides, after aligning the sequences formaximum percent identity and introducing gaps, if necessary, to achievethe maximum percent homology. N-terminal or C-terminal insertion ordeletions shall not be construed as affecting homology, and internaldeletions and/or insertions into the polypeptide sequence of less thanabout 30, less than about 20, or less than about 10 amino acid residuesshall not be construed as affecting homology. Homology or identity atthe nucleotide or amino acid sequence level can be determined by BLAST(Basic Local Alignment Search Tool) analysis using the algorithmemployed by the programs blastp, blastn, blastx, tblastn, and tblastx(Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990),Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored forsequence similarity searching. The approach used by the BLAST program isto first consider similar segments, with and without gaps, between aquery sequence and a database sequence, then to evaluate the statisticalsignificance of all matches that are identified, and finally tosummarize only those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul (1994), Nature Genetics 6, 119-129.The search parameters for histogram, descriptions, alignments, expect(i.e., the statistical significance threshold for reporting matchesagainst database sequences), cutoff, matrix, and filter (low complexity)can be at the default settings. The default scoring matrix used byblastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff(1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended forquery sequences over 85 in length (nucleotide bases or amino acids).

For blastn, designed for comparing nucleotide sequences, the scoringmatrix is set by the ratios of M (i.e., the reward score for a pair ofmatching residues) to N (i.e., the penalty score for mismatchingresidues), wherein the default values for M and N can be +5 and −4,respectively. Four blastn parameters can be adjusted as follows: Q=10(gap creation penalty); R=10 (gap extension penalty); wink=1 (generatesword hits at every wink position along the query); and gapw=16 (sets thewindow width within which gapped alignments are generated). Theequivalent Blastp parameter settings for comparison of amino acidsequences can be: Q=9; R=2; wink=1; and gapw=32. A Bestfit comparisonbetween sequences, available in the GCG package version 10.0, can useDNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extensionpenalty), and the equivalent settings in protein comparisons can beGAP=8 and LEN=2.

Thus, when referring to the polypeptide or nucleic acid sequences of thepresent disclosure, included are sequence identities of at least 40%, atleast 45%, at least 50%, at least 55%, of at least 70%, at least 65%, atleast 70%, at least 75%, at least 80%, or at least 85%, for example atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or about 100%sequence identity with the full-length polypeptide or nucleic acidsequence, or to fragments thereof comprising a consecutive sequence ofat least 100, at least 125, at least 150 or more amino acid residues ofthe entire protein; variants of such sequences, e.g., wherein at leastone amino acid residue has been inserted N- and/or C-terminal to, and/orwithin, the disclosed sequence(s) which contain(s) the insertion andsubstitution. Contemplated variants can additionally or alternatelyinclude those containing predetermined mutations by, e.g., homologousrecombination or site-directed or PCR mutagenesis, and the correspondingpolypeptides or nucleic acids of other species, including, but notlimited to, those described herein, the alleles or other naturallyoccurring variants of the family of polypeptides or nucleic acids whichcontain an insertion and substitution; and/or derivatives wherein thepolypeptide has been covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid which contains the insertion andsubstitution (for example, a detectable moiety such as an enzyme).

The term “native” is used herein to refer to nucleic acid sequences oramino acid sequences as they naturally occur in the host, organism, orvirus. The term “non-native” is used herein to refer to nucleic acidsequences or amino acid sequences that do not occur naturally in thehost, organism, or virus. A nucleic acid sequence or amino acid sequencethat has been removed from a cell or virus, subjected to laboratorymanipulation, and introduced or reintroduced into a host cell or virusis considered “non-native.” Synthetic or partially synthetic genesintroduced into a host cell or virus are “non-native.” Non-native genesfurther include genes endogenous to the virus operably linked to one ormore heterologous regulatory sequences that have been recombined intothe host genome.

A “recombinant” or “engineered” nucleic acid molecule is a nucleic acidmolecule that has been altered through human manipulation. Asnon-limiting examples, a recombinant nucleic acid molecule includes anynucleic acid molecule that: 1) has been partially or fully synthesizedor modified in vitro, for example, using chemical or enzymatictechniques (e.g., by use of chemical nucleic acid synthesis, or by useof enzymes for the replication, polymerization, digestion(exonucleolytic or endonucleolytic), ligation, reverse transcription,transcription, base modification (including, e.g., methylation),integration or recombination (including homologous and site-specificrecombination) of nucleic acid molecules); 2) includes conjoinednucleotide sequences that are not conjoined in nature, 3) has beenengineered using molecular cloning techniques such that it lacks one ormore nucleotides with respect to the naturally occurring nucleic acidmolecule sequence, and/or 4) has been manipulated using molecularcloning techniques such that it has one or more sequence changes orrearrangements with respect to the naturally occurring nucleic acidsequence. As non-limiting examples, a cDNA is a recombinant DNAmolecule, as is any nucleic acid molecule that has been generated by invitro polymerase reaction(s), or to which linkers have been attached, orthat has been integrated into a vector, such as a cloning vector orexpression vector.

The term “recombinant protein” as used herein refers to a proteinproduced by genetic engineering.

When applied to organisms or viruses, the term recombinant, engineered,or genetically engineered refers to organisms or viruses that have beenmanipulated by introduction of a heterologous or exogenous (e.g.,non-native) recombinant nucleic acid sequence into the organism orvirus, and includes, without limitation, gene knockouts, targetedmutations, and gene replacement, promoter replacement, deletion, orinsertion, or transfer of a nucleic acid molecule, e.g., a transgene,synthetic gene, promoter, or other sequence into the organism or virus.Recombinant or genetically engineered organisms or viruses can also beorganisms or viruses into which constructs for gene “knock down” havebeen introduced. Such constructs include, but are not limited to, one ormore guide RNAs, RNAi, microRNA, shRNA, siRNA, antisense, and ribozymeconstructs. Also included are organisms or viruses whose genomes havebeen altered by the activity of Cas nucleases, meganucleases, or zincfinger nucleases. An exogenous or recombinant nucleic acid molecule canbe integrated into the recombinant/genetically engineered viral ororganism's genome or in other instances are not integrated into therecombinant/genetically engineered viral or organism's genome. As usedherein, “recombinant virus” or “recombinant host cell” includes progenyor derivatives of the recombinant virus of the disclosure. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny or derivatives maynot, in fact, be identical to the parent cell, but are still includedwithin the scope of the term as used herein.

The term “engineering step” as used herein refers to the execution ofany engineering method disclosed herein or known in the art. Forexample, and “engineering step” can be a single round of an engineeringmethod of interest, such as, for example, a single round of the hereindisclosed in vitro engineering method, a single PCR-mediatedmutagenesis, or a single ligation reaction joining two pieces of DNAtogether. Likewise, “iterative engineering steps” refers to executing anengineering method two or more consecutive times.

The term “heterologous” when used in reference to a polynucleotide, agene, a nucleic acid, a polypeptide, or an enzyme, refers to apolynucleotide, gene, a nucleic acid, polypeptide, or an enzyme that isnot derived from the host species. For example, “heterologous gene” or“heterologous nucleic acid sequence” as used herein, refers to a gene ornucleic acid sequence from a different species than the species of thehost organism or virus it is introduced into. When referring to a generegulatory sequence or to an auxiliary nucleic acid sequence used formanipulating expression of a gene sequence (e.g. a 5′ untranslatedregion, 3′ untranslated region, poly A addition sequence, intronsequence, splice site, ribosome binding site, internal ribosome entrysequence, genome homology region, recombination site, etc.) or to anucleic acid sequence encoding a protein domain or protein localizationsequence, “heterologous” means that the regulatory or auxiliary sequenceor sequence encoding a protein domain or localization sequence is from adifferent source than the gene with which the regulatory or auxiliarynucleic acid sequence or nucleic acid sequence encoding a protein domainor localization sequence is juxtaposed in a genome, chromosome orepisome. Thus, a promoter operably linked to a gene to which it is notoperably linked to in its natural state (for example, in the genome of anon-genetically engineered organism or virus) is referred to herein as a“heterologous promoter,” even though the promoter may be derived fromthe same species (or, in some cases, the same organism or virus) as thegene to which it is linked. Similarly, when referring to a proteinlocalization sequence or protein domain of an engineered protein,“heterologous” means that the localization sequence or protein domain isderived from a protein different from that into which it is incorporatedby genetic engineering.

“Regulatory sequence”, “regulatory element”, or “regulatory elementsequence” refers to a nucleotide sequence located upstream (5′), within,or downstream (3′) of a coding sequence. Transcription of the codingsequence and/or translation of an RNA molecule resulting fromtranscription of the coding sequence are typically affected by thepresence or absence of the regulatory sequence. These regulatory elementsequences may comprise promoters, cis-elements, enhancers, terminators,or introns. Regulatory elements may be isolated or identified fromUnTranslated Regions (UTRs) from a particular polynucleotide sequence.Any of the regulatory elements described herein may be present in achimeric or hybrid regulatory expression element. Any of the regulatoryelements described herein may be present in a recombinant construct ofthe present invention.

The terms “promoter”, “promoter region”, or “promoter sequence” refer toa nucleic acid sequence capable of binding RNA polymerase to initiatetranscription of a gene in a 5′ to 3′ (“downstream”) direction. A geneis “under the control of” or “regulated by” a promoter when the bindingof RNA polymerase to the promoter is the proximate cause of said gene'stranscription. The promoter or promoter region typically provides arecognition site for RNA polymerase and other factors necessary forproper initiation of transcription. A promoter may be isolated from the5′ untranslated region (5′ UTR) of a genomic copy of a gene.Alternatively, a promoter may be synthetically produced or designed byaltering known DNA elements. Also considered are chimeric promoters thatcombine sequences of one promoter with sequences of another promoter.Promoters may be defined by their expression pattern based on, forexample, metabolic, environmental, or developmental conditions. Apromoter can be used as a regulatory element for modulating expressionof an operably linked transcribable polynucleotide molecule, e.g., acoding sequence. Promoters may contain, in addition to sequencesrecognized by RNA polymerase and, preferably, other transcriptionfactors, regulatory sequence elements such as cis-elements or enhancerdomains that affect the transcription of operably linked genes. A “viralpromoter” is a native or non-native promoter that initiatestranscription of one or more genes located within a viral genome.

The term “constitutive” promoter as used herein, refers to a promoterthat is active under most environmental and developmental conditions. Aconstitutive promoter is active regardless of external environment, suchas light and culture medium composition. In some examples, aconstitutive promoter is active in the presence and in the absence of anutrient. For example, a constitutive promoter may be a promoter that isactive (mediates transcription of a gene to which it is operably-linked)under conditions of nitrogen depletion as well as under conditions inwhich nitrogen is not limiting (nitrogen replete conditions). Incontrast, an “inducible” promoter is a promoter that is active inresponse to particular environmental conditions, such as the presence orabsence of a nutrient or regulator, the presence of light, etc.

The term “operably linked,” as used herein, denotes a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide sequence such that thecontrol sequence directs or regulates the expression of the codingsequence of a polypeptide and/or functional RNA). Thus, a promoter is inoperable linkage with a nucleic acid sequence if it can mediatetranscription of the nucleic acid sequence. When introduced into a hostcell, an expression cassette can result in transcription and/ortranslation of an encoded RNA or polypeptide under appropriateconditions. Antisense or sense constructs that are not or cannot betranslated are not excluded by this definition. In the case of bothexpression of transgenes and suppression of endogenous genes (e.g., byantisense or RNAi) one of ordinary skill will recognize that theinserted polynucleotide sequence need not be identical, but may be onlysubstantially identical to a sequence of the gene from which it wasderived. As explained herein, these substantially identical variants arespecifically covered by reference to a specific nucleic acid sequence.

The term “selectable marker” or “selectable marker gene” as used hereinincludes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the selection of cells that are transfected ortransformed with a nucleic acid construct of the invention. The term mayalso be used to refer to gene products that effectuate said phenotypes.Nonlimiting examples of selectable markers include: 1) genes conferringresistance to antibiotics such as amikacin (aphA6), ampicillin(amp^(R)), blasticidin (bls, bsr, bsd), bleomicin or phleomycin(ZEOCIN™) (ble), chloramphenicol (cat), emetine (RBS14p or cry1-1),erythromycin (ermE), G418 (GENETICIN™) (neo), gentamycin (aac3 oraacC4), hygromycin B (aphIV, hph, hpt), kanamycin (nptII), methotrexate(DHFR mtx^(R)), penicillin and other β-lactams (β-lactamases),streptomycin or spectinomycin (aadA, spec/strep), and tetracycline(tetA, tetM, tetQ); 2) genes conferring tolerance to herbicides such asaminotriazole, amitrole, andrimid, aryloxyphenoxy propionates,atrazines, bipyridyliums, bromoxynil, cyclohexandione oximes dalapon,dicamba, diclfop, dichlorophenyl dimethyl urea (DCMU), difunone,diketonitriles, diuron, fluridone, glufosinate, glyphosate, halogenatedhydrobenzonitriles, haloxyfop, 4-hydroxypyridines, imidazolinones,isoxasflutole, isoxazoles, isoxazolidinones, miroamide B,p-nitrodiphenylethers, norflurazon, oxadiazoles, m-phenoxybenzamides,N-phenyl imides, pinoxadin, protoporphyrionogen oxidase inhibitors,pyridazinones, pyrazolinates, sulfonylureas, 1,2,4-triazol pyrimidine,triketones, or urea; acetyl CoA carboxylase (ACCase); acetohydroxy acidsynthase (ahas); acetolactate synthase (als, csr1-1, csr1-2, imr1,imr2), aminoglycoside phosphotransferase (apt), anthranilate synthase,bromoxynil nitrilase (bxn), cytochrome P450-NADH-cytochrome P450oxidoreductase, dalapon dehalogenase (dehal), dihydropteroate synthase(sul), class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),class II EPSPS (aroA), non-class I/II EPSPS, glutathione reductase,glyphosate acetyltransferase (gat), glyphosate oxidoreductase (gox),hydroxyphenylpyruvate dehydrogenase, hydroxyphenylpyruvate dioxygenase(hppd), isoprenyl pyrophosphate isomerase, lycopene cyclase,phosphinothricin acteyl transferase (pat, bar), phytoene desaturase(crtl), prenyl transferase, protoporphyrin oxidase, the psbA photosystemII polypeptide (psbA), and SMM esterase (SulE) superoxide dismutase(sod); 3) genes that may be used in auxotrophic strains or to conferother metabolic effects, such as arg7, his3, hisD, hisG, lysA, manA,metE, nitl, trpB, ura3, xylA, a dihydrofolate reductase gene, amannose-6-phosphate isomerase gene, a nitrate reductase gene, or anornithine decarboxylase gene; a negative selection factor such asthymidine kinase; or toxin resistance factors such as a 2-deoxyglucoseresistance gene.

A “reporter gene” is a gene encoding a protein that is detectable or hasan activity that produces a detectable product. A reporter gene canencode a visual marker or enzyme that produces a detectable signal, suchas cat, lacZ, uidA, xylE, an alkaline phosphatase gene, an α-amylasegene, an α-galactosidase gene, a β-glucuronidase gene, a β-lactamasegene, a horseradish peroxidase gene, a luciferin/luciferase gene, anR-locus gene, a tyrosinase gene, or a gene encoding a fluorescentprotein, including but not limited to a blue, cyan, green, red, paprikaor yellow fluorescent protein, a photoconvertible, photoswitchable, oroptical highlighter fluorescent protein, or any of variant thereof,including, without limitation, codon-optimized, rapidly folding,monomeric, increased stability, and enhanced fluorescence variants.

The term “RNA-guided nuclease” or “RNA-guided endonuclease” as usedherein refers to a nucleic acid-cleaving enzyme that is guided to thecleavage target site by one or more guiding RNAs. Non-limiting examplesof RNA-guided nucleases include Cas9, Cpf1, C2c1, C2c2, and C2c3.

The term “terminator” or “terminator sequence” or “transcriptionterminator” as used herein refers to a regulatory section of geneticsequence that causes RNA polymerase to cease transcription.

The terms “introduction into a host cell” and “transformation” as usedherein refers to the introduction of one or more exogenous nucleic acidsequences or polynucleotides into a host cell or organism by using oneor more physical, chemical, or biological methods. Physical and chemicalmethods of transformation (i.e., “transfection”) include, by way ofnon-limiting example, electroporation, particle bombardment, chemicalinduced competency, and liposome delivery. Biological methods oftransformation (i.e., “transduction”) include transfer of DNA usingviruses or microbes (e.g., Agrobacterium).

As used herein, to “design” a genome refers to determining the desirednucleic acid sequence of the final genome of interest. The design can beinformed by basic knowledge, literature sources, experimental data, orany combination thereof.

As used herein, “recombinant” or “engineered” when referring to anucleic acid molecule, protein, viral particle, or combination thereof,means a non-naturally occurring nucleic acid molecule, protein, viralparticle, or combination thereof generated through human manipulation.As non-limiting examples, a recombinant or engineered nucleic acidmolecule includes any nucleic acid molecule that: 1) has been partiallyor fully synthesized or modified in vitro, for example, using chemicalor enzymatic techniques (e.g., by use of chemical nucleic acidsynthesis, or by use of enzymes for the replication, polymerization,digestion (exonucleolytic or endonucleolytic), ligation, reversetranscription, transcription, base modification (including, e.g.,methylation), integration or recombination (including homologous andsite-specific recombination) of nucleic acid molecules); 2) includesconjoined nucleotide sequences that are not conjoined in nature, 3) hasbeen engineered using molecular cloning techniques such that it lacksone or more nucleotides with respect to the naturally occurring nucleicacid molecule sequence, and/or 4) has been manipulated using molecularcloning techniques such that it has one or more sequence changes orrearrangements with respect to the naturally occurring nucleic acidsequence. As non-limiting examples, a cDNA is a recombinant DNAmolecule, as is any nucleic acid molecule that has been generated by invitro polymerase reaction(s), or to which linkers have been attached, orthat has been integrated into a vector, such as a cloning vector orexpression vector. A recombinant or engineered RNA or protein is onethat is transcribed or translated, respectively, from a recombinant orengineered nucleic acid molecule. A recombinant or engineered viralparticle or virus is one that is generated from an engineered viralsequence or viral genome.

The term “viral genome” refers to the complete genetic complementcontained in one or more DNA or RNA molecules in a viral particle,including genes and non-coding sequences. The term “engineered viralgenome” refers to a non-naturally occurring viral genome that is theresult of human manipulation and is able to produce non-naturallyoccurring viral particles upon introduction into a compatible host cell.

The term “viral nucleic acid” refers to a nucleic acid comprising asequence derived from a viral genome. The “viral nucleic acid” maycomprise a whole viral genome or a portion of a viral genome. Viralnucleic acids may encode amino acid sequences comprising viral proteins.In some instances, complete, mature protein or polypeptide sequencesencoded by a given viral open reading frame may not be defined orcharacterized. Amino acid sequences provided herein that are encoded byviral nucleic acid sequences that may include site suitable for mutation(such as alteration, deletion, or replacement) or insertion ofheterologous sequences can be disclosed herein as encoding amino acidsequences that may comprise all or a portion of a viral polypeptide orprotein.

The terms “viral particle” and “virion” refer to the independent form avirus exists in while not inside an infected cell or in the process ofinfecting a cell. These viral particles (virions), consist of either aDNA or RNA genome surrounded by a protein coat called a capsid. Somevirions also have an additional lipid envelope either within or externalto the capsid protein coat. The terms “viral particle”, “virion”, and“virus” can be used interchangeably.

The term “viral property” as used herein refers to any aspect of thevirus replication or life cycle or an aspect that results from the viralreplication or life cycle. As used herein, “viral property” often refersto properties that can be altered or engineered through humanintervention to achieve a desired outcome. Non-limiting examples ofviral properties include host range, viral lytic cycle, adsorption,attachment, injection, replication and assembly, lysis, burst size,immune evasion, immune stimulation, immune deactivation, biofilmdispersion, bacterial phage resistance, bacterial antibioticsensitization, modulation of virulence factors, and targeted host genomedigestion or editing. In some aspects, improved property or improvedproperties and improved viral property or improved viral properties areused interchangeably.

The terms “bacteriophage” and “phage” can be used interchangeably andrefer to a virus that infects bacteria.

CRISPR Systems

CRISPRs (clustered regularly interspaced short palindromic repeats) areDNA loci containing short repetitions of base sequences. Each repetitionis followed by short segments of “spacer DNA” from previous exposures tomobile genetic elements. CRISPRs are found in approximately 40% ofsequenced bacteria genomes and 90% of sequenced archaea. CRISPRs areoften associated with CRISPR-associated (cas) genes that code forproteins related to CRISPR function. The CRISPR-Cas system is aprokaryotic immune system that confers resistance to foreign geneticelements such as plasmids and phages and provides a form of acquiredimmunity. CRISPR spacers encode small crRNAs which sequence specificallyguide Cas endonucleases to target sequences and cut these exogenousgenetic elements in a manner analogous to RNAi in eukaryotic organisms.

Type II CRISPR-Cas systems have been used for gene editing and generegulation in many species. These systems are especially useful becausethey require only a single Cas endonuclease (Cas9) and a targetingcrRNA. In natural systems the endonuclease Cas9 requires twoindependently transcribed RNAs for activity, however, these two RNAs canalso be covalently linked to form a single chimeric gRNA. By deliveringthe Cas9 protein and appropriate gRNAs into a cell, the organism'sgenome can be cut at any desired location. CRISPR-Cas systems constitutean RNA-guided defense system which protects against viruses, plasmids,and other mobile genetic elements. This defensive pathway has threesteps. First, a copy of the invading nucleic acid is integrated into theCRISPR array. Next, the CRISPR array is transcribed into a large CRISPRtranscript and subsequently processed into mature crRNAs. The crRNAs arethen incorporated into effector complexes, where the crRNA guides thecomplex to the invading nucleic acid and the Cas proteins degrade thisnucleic acid. As stated above native type II CRISPR-Cas systems requireboth a trans-activating crRNA (tracrRNA) and pre-crRNA to enable Cas9activation. The tracrRNA is complementary to and base pairs with apre-crRNA forming an RNA duplex. This is cleaved by RNase III, anRNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid. This hybridacts as a guide for the Cas9 endonuclease, which cleaves the invadingnucleic acid generating a double-strand break in the invasive DNA toprotect the host cell. Cas9-mediated cleavage is strictly dependent onthe presence of a protospacer adjacent motif (PAM) in the target nucleicacid. The ability to program Cas9 for cleavage at specific sites definedby guide RNAs has led to its adoption as a versatile platform for genomeengineering and gene regulation. This method of genome engineering hasbeen described in U.S. Patent Application Publication Nos. 2014/0068797,published on Mar. 6, 2014, 2014/0170753, published Jun. 19, 2014, and2014/0273037 and 2014/0273226, both of which published on Sep. 18, 2014,all of which are incorporated by reference.

Other programmable CRISPR-Cas systems that can be used for genomicengineering have been described, including the Cpf1, C2c1, C2c2, andC2c3 systems. The Cpf1 system is a Type V CRISPR system and mediatessticky-end DNA cleavage through a single targeting guide RNA (Zetsche etal., Cell (2015) 163, 1-13) (incorporated by reference). C2c1 and C2c3are both Type V CRISPR systems, while C2c2 is proposed to be a Type VICRISPR system (Shmakov et al., Molecular Cell (2015) 60, 1-13)(incorporated by reference).

DNA Assembly

There are various methods known in the art for assembly of DNA duringgenetic engineering. A two-step thermocycler-based method was used toassemble portions of the M. genitalium genome, as described in Gibson,D. G., et al., “Complete chemical synthesis, assembly, and cloning of aMycoplasma genitalium genome.” Science (2008) 319:1215-1220(incorporated by reference) and PCT publication WO2009/103027(incorporated by reference). Another approach is described by Li, M. Z.,et al., Nature Meth. (2007) 4:251-256 (incorporated by reference). Asingle-step method of assembly employing T7 5′ exonuclease andsingle-stranded DNA binding protein is disclosed in PCT publicationWO2006/021944 (incorporated by reference). Combinatorial techniques forassembly of chemical compounds for use in high throughput screening isby now well established. In addition, gene shuffling techniques in whichcoding sequences are randomly fragmented and reannealed have beenpracticed for a number of years. For instance, protocols to createlibraries of chimeric gene fragments are described in Meyer, M., et al,“Combinatorial Recombination of Gene Fragments to Construct a Library ofChimeras” Current Protocols in Protein Science (2006) 26.2.1-26.2.17;McKee, A. E., et al., JBEI abstract. Techniques for assembling variouscomponents into complete or minimal genomes have been established. Forexample, U.S. Patent Publication 2000/0264688 (incorporated byreference), published Nov. 15, 2007, describes methods for constructinga synthetic genome by generating and assembling cassettes comprisingportions of the genome. A stepwise hierarchical method to assemblenucleic acids is described in U.S. Patent Publication No. 2007/004041(incorporated by reference), published Jan. 4, 2007.

Further, a one-vessel method for the assembly of DNA is described inU.S. Patent Application Publication Nos. 2010/0035768 and 2012/0053087published Feb. 11, 2010 and Mar. 1, 2012 respectively, both of which areincorporated by reference. This method has been termed the GibsonAssembly method and allows for the successful assembly of multiple DNAfragments, regardless of fragment length or end compatibility. TheGibson Assembly reaction is carried out in a single-tube underisothermal conditions using three enzymatic activities: a 5′ exonucleasegenerates long overhangs, a polymerase fills in the gaps of the annealedsingle strand regions, and a DNA ligase seals the nicks of the annealedand filled-in gaps. This method has been widely adopted and is a majorworkhorse of synthetic biology projects worldwide. Applying thismethodology, the 16.3 kb mouse mitochondrial genome was assembled from600 overlapping 60-mers. In combination with in vivo assembly in yeast,Gibson Assembly was used to synthesize the 1.1 Mbp Mycoplasma mycoidesgenome. The synthesized genome was transplanted to a M. capricolumrecipient cell, creating new self-replicating M. mycoides cells. The 5′exonuclease activity chews back the 5′ end sequences and exposes thecomplementary sequence for annealing. The polymerase activity then fillsin the gaps on the annealed regions. A DNA ligase then seals the nickand covalently links the DNA fragments together. The overlappingsequence of adjoining fragments is much longer than those used in GoldenGate Assembly, and therefore results in a higher percentage of correctassemblies.

Viruses

A virus is an ultramicroscopic and metabolically inert infectious agentthat replicates only inside the cells of living hosts. Viruses caninfect all types of life forms, including animals, plants, fungi, algae,bacteria, and archaea. While not inside an infected cell or in theprocess of infecting a cell, viruses exist in the form of independentparticles. These viral particles (virions), consist of either a DNA orRNA genome surrounded by a protein coat called a capsid. Some virionsalso have an additional lipid envelope either within or external to thecapsid protein coat.

There are two viral replication cycles, however, the terminology variesbetween prokaryotic and eukaryotic viral fields. Latent or lysogenicviruses integrate viral genetic material into the host cell's genome orform an episomal replicon. When the host cell replicates, the viralgenetic material is also copied and continues to segregate with the hostgenome until the initiation of viral production. The initiation of viralproduction and cell death are markers of the lytic or virulent cycle.During the lytic cycle, the viral genome replicated separately from thehost genome and hijacks the cell's replication and translation machineryin order to generate more viruses. Once enough viruses have accumulated,specialized viral proteins dissolve the host cell wall and/or membrane.The host cell bursts due to high internal osmotic pressure, a processcalled lysis. This releases the progeny viruses into the environmentwhere they can infect other cells and repeat the process. Virulentviruses are those that do not enter into a latent or lysogenic state,but instead replicate only through hijacking the host cell machinery (incontrast to temperate viruses, which do enter into a latent state).

Viral Mutation Studies

Viral mutation studies, as used herein, refers to rapid evolution,adaptation, and/or random or directed mutagenesis studies and the termscan be used interchangeably. Evolution and/or adaptation studiesinvolves selection of viruses for specific traits or under specificconditions. These methods are particularly useful for viruses due to thenaturally high mutation rate inherent in viral replication which leadsto a lot of viral diversity. For example, strains could be evolved underconditions of high temperature to observe the molecular changes thatfacilitate survival and reproduction under those conditions. Asnon-limiting examples, virus or bacteriophage experiment evolution oradaptation can be used to select for variants with changes in hostrange, viral lytic cycle, adsorption, attachment, injection, replicationand assembly, lysis, burst size, immune evasion, immune stimulation,immune deactivation, biofilm dispersion, bacterial phage resistance,bacterial antibiotic sensitization, modulation of virulence factors, ortargeted host genome digestion or editing. Non-limiting examples ofviral evolution or adaptation experiments include co-infection,co-evolution, or co-transformation experiments. Co-infection refers tomore than one virus infecting the same host at the same time, whichoften results in the exchange of genes between the two or more viruses.Co-evolution refers to the study in which recombination between two ormore viruses or bacteriophage occurs within a permissive ornon-permissive host that results in the assembly of a new virus orbacteriophage with different viral properties, such as, for example,wider host range. Co-transformation refers to when two naked genomes aretransformed together in a permissive or non-permissive strain. Any ofthese evolution or adaptation studies can be performed in a permissive(susceptible) or non-permissive (resistant) host. These types ofexperiments often involve passaging the virus multiple times in theselected host in the absence or presence of one or more other selectedviruses. The viruses will acquire mutations that lead to multiplevariants. Throughout the passaging, certain variants will be enrichedbased on the passaging and selection conditions.

Mutagenesis can be by any method, for example insertional mutagenesis,chemical mutagenesis, irradiation with gamma or ultraviolet radiation,or PCR-mediated mutagenesis. Methods for generating mutants or variantsof genomic sequences are well-known. For example, gamma irradiation, UVirradiation, and treatment with any of a large number of possiblechemical mutagens (e.g., 5-bromo deoxyuridine, ethyl methane sulfonate(EMS), methyl methane sulfonate (MMS), diethylsulfate (DES),nitrosoguanidine (NTG), ICR compounds, etc.) or treatment with compoundssuch as enediyne antibiotics that cause chromosome breakage (e.g.,bleomycin, adriamycin, neocarzinostatin) are methods that have beenemployed for mutagenesis of algae, fungi, and chytrids (see, forexample, U.S. Pat. No. 8,232,090; US Patent Application 20120088831; USPatent Application 20100285557; US Patent Application 20120258498). Alarge number of chemical mutagens are known in the art including but notlimited to, intercalating agents, alkylating agents, deaminating agents,base analogs. Intercalating agents include, as nonlimiting examples, theacridine derivatives or the phenanthridine derivatives such as ethidiumbromide (also known as 2,7-diamino-10-ethyl-6-phenylphenanthridiumbromide or 3,8-diamino-5-ethyl-6-phenylphenantridinium bromide).Nonlimiting examples of alkylating agents include nitrosoguanidinederivatives (e.g., N-methyl-N′-nitro-nitrosoguanidine), ethylmethanesulfonate (EMS), ethyl ethanesulfonate, diethylsulfate (DES),methyl methane sulfonate (MMS), nitrous acid, or HNO₂, and the nitrogenmustards or ICR compounds. Nonlimiting examples of base analogs that canbe used as mutagens include the compound 5-bromo-uracil (also known asdeoxynucleoside 5-bromodeoxyuridine), 5-bromo deoxyuridine, and2-aminopurine. PCR-based mutagenesis methods are well known in the artand often comprise reaction conditions and/or a DNA polymerase thatincreases the error rate throughout PCR-amplification.

Mutagenesis can additionally or alternately include introduction ofexogenous nucleic acid molecules directly into the viral genome or intothe host cell for subsequent recombination into the viral genome ofinterest. For example, an exogenous nucleic acid molecule introducedinto the host cell can integrate into a viral genetic locus by random ortargeted integration, affecting expression of genes into which theforeign DNA inserts or genes that are proximal to foreign DNA insertedinto the genome (e.g., U.S. Pat. Nos. 7,019,122; 8,216,844). Typically,the introduced nucleic acid molecule includes a selectable marker genefor selection of transformants that have integrated the exogenousnucleic acid molecule construct. The exogenous nucleic acid molecule insome embodiments can include a transposable element or a componentthereof, such as, for example, inverted repeats that can be recognizedby a transposase and/or a gene encoding a transposase, or the exogenousnucleic acid molecule can be based at least in part on a virus, such asan integrating virus.

For random insertional mutagenesis, a construct preferably includes aselectable marker that can be used to select for transformants having anintegrated construct, and optionally can also serve as a segregationmarker and molecular tag for isolation and identification of a geneinterrupted by the integrated selectable marker gene. Selective markersare not limited to antibiotic resistance genes but also include any genethat may provide a growth advantage to a virus (both genes ofestablished and hypothetical function). Alternatively, a specificgenetic locus may be targeted. The construct for gene disruption caninclude, for example, a selectable marker gene flanked by sequences fromthe genetic locus of interest, e.g., at least a portion of the gene thatencodes a regulator, and, optionally, additional genomic sequencessurrounding the gene. Such flanking sequences can comprise, for example,at least 50 nucleotides, at least 100 nucleotides, at least 500nucleotides, or at least 1 kilobase of genomic sequence.

The collection of viral variants can be generated by any of the abovementioned methods, other methods well known in the art, or anycombination thereof. The collection of variants can then be screened forthe desired phenotype. Isolated viruses with the desired phenotype/s canbe subjected to additional rounds of mutation studies. Isolated virusesdisplaying the desired properties or phenotypes can additionally oralternatively be sequenced in order to identify the genetic mutationresponsible for the desired property or phenotype. These identifiedgenetic lesions can be confirmed by recapitulating the mutation in aclean reference background and testing for the desired property orphenotype.

Viral Payloads

Lytic Enzymes

A “lytic enzyme” includes any bacterial cell wall lytic enzyme thatkills one or more bacteria under suitable conditions and during arelevant time period. Examples of lytic enzymes include, withoutlimitation, various cell wall amidases. A lytic enzyme can be abacteriophage lytic enzyme, which refers to a lytic enzyme extracted orisolated from a bacteriophage or a synthesized lytic enzyme with asimilar protein structure that maintains a lytic enzyme functionality.

A lytic enzyme is capable of specifically cleaving bonds that arepresent in the peptidoglycan of bacterial cells to disrupt the bacterialcell wall. It is also currently postulated that the bacterial cell wallpeptidoglycan is highly conserved among most bacteria, and cleavage ofonly a few bonds may disrupt the bacterial cell wall. Examples of lyticenzymes that cleave these bonds are muramidases, glucosaminidases,endopeptidases, or N-acetyl-muramoyl-L-alanine amidases. Fischetti etal. (1974) reported that the C1 streptococcal phage lysin enzyme was anamidase. Garcia et al. (1987, 1990) reported that the Cpl lysin from aS. pneumoniae from a Cp-1 phage is a lysozyme. Caldentey and Bamford(1992) reported that a lytic enzyme from the Pseudomonas phage 16 is anendopeptidase, splitting the peptide bridge formed bymelo-diaminopimilic acid and D-alanine. The E. coli phage T1 and T6lytic enzymes are amidases as is the lytic enzyme from Listeria phage(ply) (Loessner et al., 1996). There are also other lytic enzymes knownin the art that are capable of cleaving a bacterial cell wall.

A lytic enzyme genetically encoded for by a bacteriophage includes apolypeptide capable of killing a host bacterium, for instance by havingat least some cell wall degrading or cell wall synthesis inhibitingactivity against the host bacteria. The polypeptide may have a sequencethat encompasses native lytic enzymes and variants thereof. Thepolypeptide may be isolated from a variety of sources, such as from abacteriophage (“phage”), or prepared by recombinant or syntheticmethods. The polypeptide may, for example, comprise a choline-bindingportion at the carboxyl terminal side and may be characterized by anenzyme activity capable of cleaving cell wall peptidoglycan (such asamidase activity to act on amide bonds in the peptidoglycan) at theamino terminal side. Lytic enzymes have been described which includemultiple enzyme activities, for example two enzymatic domains, such asPlyGBS lysin. Further, other lytic enzymes have been describedcontaining only a catalytic domain and no cell wall binding domain.

Quorum Quenching Polypeptides

Autoinducers are small chemical signaling molecules produced and used bybacteria participating in quorum sensing. Quorum sensing allows bacteriato sense one another via the presence of autoinducers and to regulate awide variety of group-level behaviors. Such behaviors include symbiosis,virulence, motility, antibiotic production, and biofilm formation.Autoinducers come in a number of different chemical forms depending onthe species, but the effect that they have is similar in many cases,which allows genetically engineered bacteriophages to impact a widevariety of bacteria utilizing similar autoinducers. In general,Gram-negative bacteria use AHL as autoinducers, and Gram-positivebacteria use processed oligo-peptides to communicate, while autoinducer2 (AI-2) is universal for Gram-negative and Gram-positive bacteria.

AHLs produced by different species of Gram-negative bacteria vary in thelength and composition of the acyl side chain, which often contains 4 to20 carbon atoms. AHLs are capable of diffusing in and out of cells byboth passive transport and active transport mechanisms. Receptors forsensing AHLs include a number of transcriptional regulators, such asLuxR, which function as DNA binding transcription factors that canactivate diverse gene expression regulating bacterial populationbehaviors.

Autoinducers can be inhibited by quorum quenching polypeptides. Quorumquenching polypeptides can modify or degrade autoinducers to render themless active or inactive. Certain quorum quenching polypeptides areenzymes that inactivate an autoinducer (e.g., by modification ordegradation), such as the AiiA lactonase protein described herein thatcleave the lactone rings from the acyl moieties of AHLs with broad-rangesubstrate specificity for inactivating AHL from various bacteria (Wanget al. (2004) J. Biol. Chem. 279(14):136.45-51).

The herein disclosed in vitro engineering method can be employed togenerate synthetic bacteriophage engineered to encode, for example, aquorum quenching polypeptide derived from Pseudomonas aeruginosa. Thequorum quenching polypeptides can be expressed as free proteins that arereleased into the area surrounding a phage and/or bacteria, e.g., uponphage infection and lysis of the host bacteria. Equally possible, thequorum quenching polypeptides can also be expressed and activelysecreted from the bacterial host cell using methods known in the art.Similarly, quorum quenching polypeptides can be translationally fused toa bacteriophage protein, e.g., a capsid, tail, or neck protein.

Tail Fibers

The disclosure contemplates, in some embodiments, tuning bacteriophagehost range by engineering recombinant bacteriophage. In someembodiments, tuning virus host range involves engineering the virus tohave heterologous, native, non-native tail fibers, and any combinationthereof. Host cell specificity of bacteriophage can be influenced by theviral particle tail fiber(s). By altering (e.g., swapping and/ormutating) tail fibers, or portions of tail fibers, of a hostbacteriophage, the host range can be altered (e.g., expanded).

Tail fiber proteins typically contain antigenicity determinants and hostrange determinants. A heterologous tail fiber may be encoded by a set ofgenomic fragments isolated from or synthesized based upon the genome ofone type of bacteriophage. The set of tail fiber gene fragments maycontain subsets of genomic fragments isolated from or generated basedupon the genomes of several bacteriophages. For example, conservedregions of a tail fiber may be encoded by genomic fragments isolatedfrom the genome of the chassis bacteriophage, while host rangedeterminant regions may be encoded by genomic fragments isolated fromthe genome of a different type of bacteriophage.

Anti-Microbial Peptides

The disclosure contemplates, as a non-limiting example, bacteriophageengineered to express an antimicrobial peptide which is optionallysecreted by the host cell. For example, engineered bacteriophages canexpress an antimicrobial agent, such as an antimicrobial peptide (AMP)or antimicrobial polypeptide, including but not limited to naturallyoccurring peptides to prevent the development and/or propagation ofresistance of the host bacteria to the bacteriophage, and to allow forfaster and more effective killing of bacteria in bacterial infections,such as bacterial infections comprising more than one differentbacterial species.

Bacteriophages provide an attractive antimicrobial agent for eliminatingbacterial infections due to their amplification and predator-hostmechanism, e.g. by propagating in the host bacteria and then killing thebacteria as lysis occurs to release the propagated bacteriophages whichsubsequently infect and kill the surrounding bacteria by the samemechanism. The practical use of bacteriophage in eliminating bacterialinfections is stemmed by significant limitations such as (i) a verynarrow bacteria host-range both intra- and inter-species, and (ii) veryrapid development of resistance against the bacteriophage by thebacterial host population. Thus, as seems common in many areas ofscience, the theoretical outcome is difficult to achieve in real lifesituations. Therefore, while bacteriophages appear useful asantimicrobial agents in theory, in practice they have restrainedantimicrobial properties, and their use for eliminating bacterialinfections is very difficult to achieve due to the rapid development ofhost resistance to the bacteriophage. Consequently, bacteriophages havebeen ineffective at long-term elimination of the host bacteria.

Accordingly, the present disclosure contemplates antimicrobial-agentengineered bacteriophage where the bacteriophage is modified orengineered to express an antimicrobial peptide (AMP) which is optionallysecreted by the host cell. At least one, or any combination of differentantimicrobial-agent engineered bacteriophage can be used alone, or inany combination to eliminate or kill a bacterial infection. In someembodiments, an antimicrobial-agent engineered bacteriophage can be usedwith additional agents, such as other antimicrobial-agent engineeredbacteriophage, purified antimicrobial peptide(s), or small moleculeantibiotic. The antimicrobial peptide-engineered bacteriophages (orAMP-engineered bacteriophages) can encode any antimicrobial-agent knownto one of ordinary skill in the art.

In some embodiments of aspects of the invention, an antimicrobial-agentengineered bacteriophage can express and secrete an antimicrobial agentwhich is a nucleic acid, for example an antimicrobial agent whichfunctions by “gene silencing” commonly known bacterial genes known bypersons of ordinary skill in the art. A nucleic acid-based antimicrobialagent includes for example, but is not limited to, RNAinterference-inducing (RNAi) molecules, for example but are not limitedto siRNA, dsRNA, stRNA, shRNA, miRNA and modified versions thereof,where the RNA interference molecule gene silences the expression of agene expressed and important for viability (i.e. survival) of thebacteria. A nucleic acid-based antimicrobial agent can be an anti-senseoligonucleic acid, or a nucleic acid analogue, for example but are notlimited to DNA, RNA, peptide-nucleic acid (PNA), pseudo-complementaryPNA (pc-PNA), or locked nucleic acid (LNA) and the like. Alternatively,a nucleic acid-based antimicrobial agent can be a DNA or RNA, andnucleic acid analogues, for example PNA, pcPNA and LNA. A nucleic acidcan be single or double stranded, and can be selected from a groupcomprising nucleic acid encoding a protein of interest,oligonucleotides, PNA, etc. Such nucleic acid inhibitors include forexample, but are not limited to, a nucleic acid sequence encoding aprotein that is a transcriptional repressor, or an antisense molecule,or a ribozyme, or a small inhibitory nucleic acid sequence such as aRNAi, an shRNAi, an siRNA, a micro RNAi (miRNA), an antisenseoligonucleotide etc.

Antimicrobial peptides can additionally or alternatively beantibacterial enzymes. Exemplary antibacterial activities can include,but re not limited to, a lytic enzyme, an acylase, an aminopeptidase, anamylase, a carbohydrase, a carboxypeptidase, a catalase, a cellulase, achitinase, a cutinase, a cyclodextrin glycosyltransferase, adeoxyribonuclease, an esterase, an alpha-galactosidase, abeta-galactosidase, a glucoamylase, an alpha-glucosidase, abeta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, amannosidase, an oxidase, a pectinolytic enzyme, a peptidoglutaminase, aperoxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, aribonuclease, a transglutaminase, a xylanase, RNase, DNase, lysostaphin,or pore forming peptides.

Antimicrobial peptides or antimicrobial polypeptides can directlydisrupt the bacterial membrane by binding to the negatively chargedmicrobial membrane and disrupting the membrane by forming aqueouschannels, causing the lipid bilayer to fold back on itself or blanketingthe membrane to form micelles. In addition to their direct bactericidaleffects, anytimicrobial peptides and polypeptides may also activate TLRsignaling and additional immunue responses, serve as leucocytechemoattractants, increase bactericidal opsonization by invadingphagocytes, scavenge vital nutrients that bacteria need for growth andinhibit bacterial proteases, or any combination thereof.

Biosurfactants

Bacterial biofilm formation can lead to localized infections as well asdifficult to treat, and sometimes fatal, systemic infections, such asbacteremia (the presence of bacteria in the blood) and bacterial sepsis(multiple organ failure caused by the spread of bacteria or theirproducts through the bloodstream). The extracellular substances thatcomprise the biofilm matrix can act as a barrier that protects andisolates the bacteria resident within the biofilm from normalimmunological defense mechanisms, such as antibodies and phagocytes, aswell as from antimicrobial agents including antibacterial enzymes andantibiotics. The biofilm also facilitates the growth and proliferationof bacteria resident within the biofilm.

The present disclosure provides for methods of generating andcompositions of engineered viruses expressing an additional agent usedto facilitate removing or loosening the biofilm deposited on a surface.For example, the compositions can include a biosurfactant. Exemplarybiosurfactants included, but are not limited to, glycolipids,lipopeptides, depsipeptides, phospholipids, substituted fatty acids,lipopolysaccharides, surlactin, surfactin, visconsin, and rhamnolipids.

Viral Engineering

Methods of genetically engineering viral particles are laborious andlengthy due to the lack of widely applicable and targetable in vitroengineering methods. Current in vivo methods may take weeks or months tocreate modified viruses and viral vectors (Levin and Bull, Nat RevMicrobiol., 2004 February; 2(2):166-73, incorporated herein byreference). Additionally, there is toxicity inherently associated withthe manipulation of viral genomes in cells. Prior to this disclosure,efforts to develop widely applicable methods for precise in vitrogenetic engineering of viruses have been largely unsuccessful. Herein isdescribed a widely applicable process to rapidly engineer viral genomescompletely in vitro.

The herein disclosed in vitro genetic engineering systems and methodshave several advantages over existing methods of viral geneticengineering: 1) it allows simple manipulation of toxic genes/productscompletely in vitro; 2) it is rapid, i.e. can be performed in a daycompared to weeks or months for in vivo methods; 3) it allows retentionof genomic modification over most of viral genome; 4) it does notrequire host recombination pathways; 5) it is more direct and less errorprone than previous methods; and 6) it is applicable to multiple viruseswithout changes to protocol.

The present disclosure provides methods for RNA-guided nuclease mediateddigestion and in vitro assembly to site specifically engineer wholegenomes. The present disclosure significantly increases the precision,simplicity, and speed at which viral genomes can be geneticallymodified. Further, this technique overcomes the well-establisheddifficulty of manipulating often toxic virulent viral genomes insidehost cells. This completely in vitro approach also removes therequirement for a genetically tractable host strain for engineering, arequirement that prevents the manipulation of many important andinteresting viruses of Archaea, Prokaryotes, and Eukaryotes. Thisapproach does not amplify the viral genomes being manipulated and soallows retention of most viral genome modifications such as methylation.It is well established that genome modifications can have, profoundeffects on the fitness of viruses and so the retention of these genomemodifications provides a distinct advantage over other engineeringtechniques. Additionally, this technique is distinct from other methodspertaining to in vivo RNA-guided nuclease genome engineering as it doesnot center on the use of RNA-guided nuclease, such as Cas9, and gRNAsfor eukaryotic genome editing, but instead pertains to overcoming knownviral engineering problems completely in vitro.

In some aspects, the novel methods provided herein can includemodification of the viral nucleic acid or viral genome, for exampleusing an RNA-guided nuclease and assembly as disclosed herein andintroduction of the engineered viral nucleic acid or engineered viralgenome directly into a host that will produce engineered viral particlesor engineered viruses that comprise the engineered viral nucleic acid orengineered viral genome. For example, in some aspects, the methodsinclude engineering a viral nucleic acid or viral genome withoutintroducing the engineered viral nucleic acid or engineered viral genomeinto a cloning host for the purposes of amplification of the engineeredviral nucleic acid or engineered viral genome, for example, throughreplication in a vector. For example, in some methods, the engineeredviral nucleic acid or engineered viral genome is not introduced intoyeast, E. coli, or other known cloning hosts such as, but not limitedto, Bacillus or Vibrio species, prior to introduction of the engineeredviral nucleic acid or engineered viral genome into a host cell that willproduce engineered viral particles or engineered viruses.

The novel methods provided herein allow for targeted engineering of two,three, four, five, or more sites in a viral genome. The methods can beperformed entirely in vitro, allowing for the production of viralgenomes altered at multiple sites, a feat not achieved usingconventional engineering methods. Provided herein are engineered virusescomprising engineered viral nucleic acid and/or engineered viral genomesthat have two, three, four, five, or more modifications with respect tothe non-engineered viral nucleic acid or non-engineered viral genome.The two or more modifications can be an insertion, deletion,replacement, or any combination thereof. The two or more modificationscan lead to one, two, or more improved viral properties, such as anydisclosed herein. The engineered viruses can be generated entirelythrough the in vitro engineering methods disclosed herein. The in vitroengineering methods as disclosed herein result in targeted modificationsas opposed to classical or random mutagenesis. Unlike modificationsgenerated by classical or random mutagenesis, the targeted modificationscan be conveniently screened for using standard molecular geneticlaboratory methods such as PCR and/or sequencing prior to any phenotypicassays.

Also disclosed herein is a system for generating synthetic viruses withimproved viral properties (For example, see FIG. 10). The systemcomprises identifying nucleic acid sequences responsible for conferringa desired property and then incorporating those sequence changes into aselected viral genome in order to generate viral particles with improvedviral properties. The nucleic acid sequences capable of conferring adesired viral property can be identified through basic scientificknowledge, literature search, empirical testing, mutation studies, orany combination thereof. Mutation studies can include evolution studies,adaptation studies, mutagenesis studies, and/or other experimentalapproaches well known in the art. Mutagenesis studies can include ultraviolet (UV), chemical, and/or insertional mutagenesis. Insertionalmutagenesis can include transposon and/or selectable marker insertionalmutagenesis. The mutation experiments used to identify nucleic acidsequences of interest can be performed using the virus or viral genomeof the virus which will be the starting point for the in vitroengineering. Additionally or alternatively, instead of the selectedvirus or viral genome, a related or heterologous virus or viral genomecan be used in a mutation study in order to identify recombinant nucleicacid sequences to incorporate into the originally selected virus orviral genome in order to confer additional properties to the selectedvirus.

The desired properties can include one or more of host range, virallytic cycle, adsorption, attachment, injection, replication andassembly, lysis, burst size, immune evasion, immune stimulation, immunedeactivation, biofilm dispersion, bacterial phage resistance, bacterialantibiotic sensitization, modulation of virulence factors, and targetedhost genome digestion or editing, other desirable properties that wouldbe readily known by one of skill in the art, or any combination thereof.The identified nucleic acid sequences conferring the desired propertycan be incorporated into the selected viral genome using the hereindisclosed in vitro engineering method to incorporate one or more changesinto a single viral genome through one or more rounds of iterativeengineering and testing until the desired set of one or more improvedviral properties have been confirmed. The final viral genome of interestcan be a combination of naturally-derived and synthesized nucleic acidmolecules, or can be completely synthesized de novo using methodsdescribed herein and/or those known in the art. Generating viruses orviral particles with improved viral properties can involve introducingthe engineered viral genome of interest into a compatible cell, whereinthe genome is activated thereby generating viral particles or viruses.To prepare the nucleic acid molecule identified to confer a desiredproperty for incorporation into the selected viral genome, the sequenceof interest can be isolated or amplified from the viral genome fromwhich it was identified by digestion, PCR-amplification, synthesized,other methods well known in the art, or any combination thereof.Synthesized nucleic acid sequence can be chemically synthesized orassembled from chemically synthesized overlapping oligonucleotides.Additionally or alternatively, the nucleic acid molecule to beincorporated into the selected viral genome in order to confer thedesired phenotype can be a combination of naturally-derived andsynthesized nucleic acid sequences. Depending on the design of thenucleic acid molecule to be incorporated into the selected viral genome,the resulting engineered viral genome can have nucleic acid sequencesadded, deleted, replaced with alternative sequences, or any combinationthereof in order to confer the desired viral property. Methods ofdesigning nucleic acid molecules in order to alter a sequence in such away that sequences are removed, deleted, replaced, or any combinationthereof are well known to one skilled in the art. Engineered viralgenomes generated by the herein described system and methods can be usedto generate viruses or viral particles with improved viral properties.Generating viruses or viral particles with improved viral properties caninvolve introducing the engineered viral genome into a compatible cell,wherein the genome is activated thereby generating viral particles orviruses. Introducing the engineered genome into the cell can beperformed by electroporation, transformation, conjugation, contact ofthe cell with pre-packaged viral genomes, etc. or other methods wellknown in the art.

The present disclosure additionally relates to the discovery of a methodfor engineering nucleic acid in vitro using a RNA-guided endonuclease.This disclosure further relates to the improvement of viral propertiesby in vitro genetic engineering of viral nucleic acids. Specifically,the disclosure relates to the in vitro digestion of viral sequencesusing an endonucleases, such as an RNA-guided endonuclease, e.g., Cas9,followed by the assembly of a recombinant nucleic acid by the insertionof a DNA or RNA fragment(s) into the digested viral genome.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral nucleic acid comprising isolation of a viralnucleic acid; in vitro digestion of a region of the viral nucleic acidusing a RNA-guided nuclease; and assembly of a recombinant nucleic acidby the insertion of a DNA or RNA fragment into the digested viralnucleic acid. In some examples, the in vitro digestion is an RNA-guidedenzymatic digestion. In some examples, the enzymatic digestion isperformed by an RNA-guided nuclease. In some examples, the RNA-guidednuclease is Cas9, a Cas9-derived enzyme, a Cas9-related enzyme, or anypurified programmable RNA-guided nuclease. In some examples, thedigestion further comprises targeting RNAs. In some examples, thedigestion further comprises spermidine. In some examples, the targetingRNAs are gRNA, crRNA and/or tracrRNA. In some examples, followingdigestion, the RNA-guided nuclease is inactivated by standard methodssuch as exposure to heat and/or removed by standard methods, such as,for example, phenol-chloroform extraction. In some examples, heat inactivation is achieved by exposing the protein comprising solution toheat, such as, for example, at least 80° Celcius.

Any programmable RNA-guided nuclease can be used in the methods andcompositions herein, e.g., Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash,Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2,Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10,Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1,C2c2, C2c3, or homologs thereof, or modified versions thereof. Anyprogrammable CRISPR system can be used in the methods and compositionsherein, including Type I, Type II, Type III, Type IV, Type V, Type VI,or any combination thereof. The RNAi-guided nuclease can be a Cas9protein, such as a Cas9 protein of Staphylococcus pyogenes, S.thermophilus, S. pneumonia, S. aureus, or Neisseria meningitidis, asnonlimiting examples. Also considered are the cas9 proteins provided asSEQ ID NOs:1-256 and 795-1346 in U.S. Patent Application Publication No.US 2014/0068797, incorporated by reference herein in its entirety, andchimeric Cas9 proteins that may combine domains from more than one Cas9protein, as well variants and mutants of identified Cas9 proteins. Inaddition to Cas9, it would be readily recognized by one of skill in theart that any known functional equivalent would be a sufficientalternative example.

The viral particles may be archaeal-, prokaryotic-, oreukaryotic-specific viruses. For example, the virus can be one that caninfect Pseudomonas aeruginosa, E. coli, or Homo sapiens. In someexamples, the virus can be one that infects pathogen species such asthose in the genus of Acinetobacter, Clostridium, Enterobacter,Enterococcus, Escherichia, Klebsiella, Mycobacterium, Neisseria,Pseudomonas, Salmonella, Staphylococcus, or Streptococcus. In someexamples, the virus can infect archaeal species such as those in thegenus Acidianus, Aeropyrum, Haloarcula, Haloferax, Halorulbum,Methanobacterium, Pyrobaculum, Pyrococcus, Stygiolobus, Sulfolobus, orThermoproteus. In some examples, the virus can infect eukaryotic hostssuch as humans, mammals, animals, plants, algae, or fungi. The viralnucleic acid may be DNA or RNA. In some examples, the viral nucleic acidconsists of an entire viral genome, a portion of the viral genome, or asingle or multiple viral genes. In some examples, a portion of a viralgenome is subcloned into a plasmid prior to engineering.

The viral nucleic acid may be single or double (or more) digested by anRNA-guided nuclease, such as Cas9, coupled with targeting RNA(s) invitro to remove one or more nucleotides, a single gene, multiple genes,or any size genomic region or to open the DNA for insertion of a newsequence. In addition to Cas9 it is understood by one skilled in the artthat any programmable RNA-guided nuclease or other targetable DNAcleavage mechanism would suffice and would be functionally equivalent.Multiple digestions can be performed concurrently; however, it was foundthat sequential RNA-guided Cas9 digestion can increase efficiency.Further, spermidine can be added to the reaction mixture to increaseCas9 dissociation from DNA, allowing for greater availability of Cas9for enzymatic activity. The viral sequence removed by Cas9 cleavage doesnot recombine back into the genome because Cas9 is a blunt cuttingenzyme and fragments do not contain homology to insertion site.Additionally, heat deactivation of Cas9 allows for direct movement fromdigestion into assembly reactions, simplifying the protocol.

As used herein, the term “targeting RNAs” or “guiding RNAs” refers toCRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), engineeredchimeric guide RNAs (gRNAs) incorporating both crRNAs and tracrRNAs, orsingle gRNAs compatible with the chosen CRISPR system. CRISPR RNAs(crRNAs) are transcribed from a CRISPR locus, are incorporated intoeffector complexes and guide the complex to the invading nucleic acidsequences resulting in RNA-guided nuclease mediated digestion of thenucleic acid. TracrRNAs are complementary to and base pairs with apre-crRNA forming an RNA duplex required for Cas9 mediated cleavage.Hybrid gRNAs are chimeric RNAs that link the targeting crRNA with atracrRNA, allowing for the use of a single RNA for Cas9 mediateddigestion. Cas9 mediated digestion can be performed with both in vitrotranscribed crRNA-tracrRNA mixtures or with chimeric gRNAs.

The DNA or RNA insert can be obtained by any means known in the art andspecifically through in vitro synthesis, chemical synthesis, de novosynthesis, de novo assembly, amplification (PCR), enzyme mediatedliberation from plasmids, viruses, or bacteria, or any combinationthereof. In one aspect, the DNA or RNA insert is generated by theassembly of oligos or PCR with primers containing overlapping sequencesto integration site. The DNA or RNA insert can be a combination ofnaturally-derived and synthesized nucleic acids, or wholly naturally orsynthetically derived.

The assembly of the DNA or RNA insert and the digested viral nucleicacid can be performed using any method known in the art, such as invitro cloning reactions or any of the methods previously discussed. Inone aspect, the assembly of the DNA or RNA insert into the digestedviral genome is performed using the Gibson Assembly method. In oneaspect, the assembly of the DNA or RNA insert into the digested viralgenome is performed in vivo using the host cells recombinationmachinery. The assembly of the DNA or RNA insert can result in theaddition, deletion, replacement, or any combination thereof, of nucleicacid sequence. The process of designing a DNA or RNA sequence such thatassembly into the digested viral nucleic acid results in the addition,deletion, replacement, or any combination thereof of nucleic acids ofinterest are well known in the art.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising isolation of a viral nucleicacid; in vitro digestion of a region of the viral nucleic acid using aRNA-guided nuclease; and assembly of a recombinant nucleic acid by theinsertion of a DNA or RNA fragment into the digested viral nucleic acid.In some examples, the assembly is performed in vitro in a single vesselwith a mixture of components comprising (a) an isolated non-thermostable5′ to 3′ exonuclease that lacks 3′ exonuclease activity, (b) a crowdingagent, (c) an isolated thermostable non-strand-displacing DNA polymerasewith 3′ exonuclease activity, or a mixture of said DNA polymerase with asecond DNA polymerase that lacks 3′ exonuclease activity, (d) anisolated thermostable ligase, (e) a mixture of dNTPs, and (f) a suitablebuffer, under conditions that are effective for insertion of thefragment into the digested viral nucleic acid to form a recombinantnucleic acid. In some aspects, the exonuclease is a T5 exonuclease andthe contacting is under isothermal conditions, and/or the crowding agentis PEG, and/or the non-strand-displacing DNA polymerase is Phusion™ DNApolymerase or VENT® DNA polymerase, and/or the ligase is Taq ligase. Insome examples, the in vitro assembly is performed by one-step orisothermal Gibson assembly. In some examples, the in vitro assembly isperformed by two-step Gibson assembly. In some examples, the digestednucleic acid and the DNA or RNA fragment can be assembled in vitro byblunt ligation using a ligase enzyme.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising an assembly step. In someexamples, the assembly is performed in vivo in a compatible host cellusing the host cell recombination machinery. While the recombinantnucleic acid can be assembled completely in vitro utilizing purifiedenzymes as disclosed herein, this process can also be accomplishedutilizing natural or engineered recombination pathways within asusceptible host strain. In some instances, compatible host cells can beS. cerevisiae, E. coli, P. aeruginosa, B. subtilis, V. natrigens, orother organism available in the art. Transformation of purified and invitro digested viral genomes along with an insert repair fragmentharboring terminal homology regions is sufficient for some host cells toassemble a recombinant viral genome in vivo. Insert repair fragments canbe synthesized or amplified by standard techniques known in the art orcan reside within plasmids stably replicating within the chosen hostcell. This method is likely to have lower efficiency than in vitroassembly due to host cells having both homologous and non-homologous DNArepair pathways, the challenge of co-delivering sufficient quantities ofinsert and digested genome into a host cell, and the lower efficiency ofmost host homologous recombination pathways. As digested genomes alonewill not form functional viral particles and subsequent plaques withouthost-mediated recombination, the plaques obtained followingtransformation and plating can be screened by PCR for the given insertto confirm correct assembly of the desired engineered viral nucleicacid.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising an RNA-guided nuclease. Insome examples, the RNA-guided nuclease is a Type II Cas9. In someexamples, the RNA-guided nuclease is Cas9 or a Cas9 derived enzyme. Insome examples, the RNA-guided nuclease is an isolated recombinant Cas9or Cas9 derived enzyme. In some examples, there is at least onetargeting RNA. In some examples, there are two targeting RNAs. In someexamples, the targeting RNA is a chimeric guide RNA (gRNA) or a set of acrRNA and tracrRNA. In some examples, the in vitro digestion reactionuses two gRNAs. In some examples, the in vitro digestion reaction usestwo sets of crRNAs and tracrRNAs in order to, for example, target twosequences concurrently.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising an in vitro digestion step.In some examples, following digestion, the RNA-guided nuclease isinactivated by standard methods such as exposure to heat, such as atleast 80° Celcius. In some examples, following digestion, the RNA-guidednuclease is removed by phenol-chloroform extraction. In some examples,following digestion, the RNA-guided nuclease is removed by otherextraction methods well known in the art.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence that results in an engineered viralnucleic acid. In some examples, the engineered viral nucleic acid isthen transformed into a host cell. In some examples, the host cell is E.coli, P. aeruginosa, S. cerevisiae, V. natriegens, B. subtilis, or otherorganism well known in the art. In some examples, the transformation isperformed by heat shock, electroporation, biolistics, particlebombardment, conjugation, transduction, lipofection, or otherestablished method well known in the art. In some examples, theengineered viral nucleic acid is transformed into a host cell and thenagain isolated following replication. In some examples, the isolatedengineered viral nucleic acid is used as the starting viral nucleic acidfor another round of in vitro engineering, a process herein referred toas iterative in vitro engineering. In some examples, there is one roundof iterative in vitro engineering. In other examples, there is at leastone round of iterative in vitro engineering. In other examples, thereare two or more rounds of iterative in vitro engineering.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence that results in an engineered viralnucleic acid. In some examples, the engineered viral nucleic acid ispackaged into viral particles using an in vitro packaging kit that canbe commercially available. In some examples, the in vitro packaging kitis the Maxplax lambda packaging extract.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence that results in a recombinant engineeredviral nucleic acid. In some examples, the engineered viral nucleic acidimproves or alters a property of the virus compared to the referenceand/or non-engineered virus. In some examples, the improved or alteredviral property is a property such as host range, viral lytic cycle,adsorption, attachment, injection, replication and assembly, lysis,burst size, immune evasion, immune stimulation, immune deactivation,biofilm dispersion, bacterial phage resistance, bacterial antibioticsensitization, modulation of virulence factors, targeted host genomedigestion or editing, or any combination thereof. In some examples, theimprovement of a property can be an increase, decrease, or alteration ofthe property. For example, the improved viral property can be expandedor reduced host range, altered viral lytic cycle, increased or decreasedadsorption to a host cell, increased or decreased attachment to a hostcell, increased or decreased injection, increased or decreased oraltered replication and assembly, increased or decreased lysis,increased or decreased burst size, increased or decreased or alteredimmune evasion, increased or decreased or altered immune stimulation,increased or decreased or altered immune deactivation, increased ordecreased or altered biofilm dispersion, increased or decreased oraltered bacterial phage resistance, increased or decreased or alteredbacterial antibiotic sensitization, increased or decreased or alteredmodulation of virulence factors, increased or decreased or alteredtargeted host genome digestion or editing, or any combination thereof.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, increased host range. Host range is thenumber of cell types, strains, or host species a virus is able toinfect. Increase of host range is an expansion of the absolute number ofdistinct cell types, strains, or species a virus is able to infectcompared to a reference and/or non-engineered virus. In some examples,increased host range is an increase in the number of bacterial strainsor variants within a bacterial species that the virus is able to infect.The increase in host range can be an increase of at least one or morethan one strain, cell type, or species. Host range can assayed, forexample, by a standard plaque assay that is well known in the art.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, the viral lytic cycle. The viral lyticcycle is one of the two cycles of viral replication, the other being thelysogenic cycle. The lytic cycle results in the destruction of theinfected cell and the infected cell membrane. The lytic cycle comprisessix steps, which can each be individually engineered. The six steps inthe viral lytic cycle are adsorption, attachment, injection, replicationand assembly, lysis, and burst size.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, adsorption. Adsorption is the act of thevirus contacting the host cell. Viral adsorption is characterized as theaffinity of a virus for a given host cell and can be assayed by standardadsorption assays, such as those outlined by Hyman and Abedon (Methodsin Molecular Biology, 2009). Additionally, or alternatively, viraladsorption can be determined by other standard affinity assays widelyused in biochemistry to analyze receptor-ligand interactions.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, attachment. Viral attachment is when thevirus strongly attaches to the host cell. Viral attachment is anirreversible interaction between the virus and the host cell receptor.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, injection. Injection refers to viralgenome injection and is when the virus inserts its genetic material intothe host cell. Viral genome injection can be measured, as an example, bymeasurement of potassium ion efflux (Cady et al., J. Bacteriol 2012November; 194(21):5728-38; Leavitt et al., PLoS ONE, 2013 8(8): e70936,both incorporated herein by reference in their entirety).

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, replication and assembly. Viralreplication and assembly refers to the host cell building new viruses.Following viral genome injection, the host cell machinery is hijackedand viral genes are transcribed, viral proteins are translated, andviral particles are assembly comprising replicated viral genomes. Viralreplication and assembly will ultimately lead to host cell lysis,therefore, replication and assembly can be assayed monitoring the viralgrowth rate by standard plaque assay or the double agar plaque assay.Viral replication rates can additionally or alternatively be determinedby measuring burst size in a standard plaque assay, one-step curve, orby other standard viral fitness assays that are well known in the art.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, lysis. Lysis refers to host cell lysis.After replication and assembly of new virus particles, an enzyme isproduced that breaks down the host cell wall and/or cell membrane fromwithin and allows fluid to enter, which ultimately leads to host celllysis. The ability to increase or inhibit the virulent replication of avirus can increase or decrease the time it takes for a given virus tokill a host cell by lysis. Viral virulence can be assayed by analyzingthe time between infection and host cell lysis, by monitoring the viralgrowth rate by standard plaque assay or the double agar plaque assay.Additionally or alternatively, increased bacterial lysis of anengineered virus compared to a reference and/or non-engineered virus canbe determined by colony forming units (CFUs) following an assay, plaqueforming units (PFUs) number or diameter following a plaque assay, frombiofilm assays, or other standard assays that are well known in the art.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, burst size. Burst size refers to thenumber of viruses produced by an infected cell. Burst size can beassayed by standard burst size assays such as those outlined by Ellisand Delbruck (J Gen Physiol. 1939 Jan. 20; 22(3): 365-384, incorporatedherein by reference) and Delbruck (Delbruck, J. Gen. Physiol, 1940, 23;643, incorporated herein by reference)

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, immune evasion. Immune evasion is theability of a virus to avoid clearance by the innate or adaptive immunesystem. Immune evasion can be assayed by looking at the level or speedof neutralizing antibody production. Additionally, or alternatively,immune evasion can be measured by analyzing the half-life or residencytime of a given virus within an animal.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, immune stimulation. Immune stimulationis the ability of a virus to induce an immune response not normallyassociated with the wild type or non-engineered virus. This can beassayed by analyzing the immune factors produced in the presence of thevirus using standard ELISA kits, flow cytometry, histology, or othercommon immunological assays known to those skilled in the art.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, immune deactivation. Immune deactivationis the ability of a virus to decrease an immune response normallyassociated with the wild type or non-engineered virus. This can beassayed by analyzing the immune factors produced in the presence of thevirus using standard ELISA kits, flow cytometry, histology, or othercommon immunological assays known to those in the art.

In some aspects, the present disclosure provides a method forengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, biofilm dispersion. Biofilm dispersionis the ability to degrade, loosen, or increase the penetrability of abiofilm. Activities that can lead to biofilm dispersion include, but arenot limited to, exopolysaccharide (EPS) degradation, modulation ofquorum sensing molecules, and degradation of extracellular DNA or RNAwithin a biofilm or bacterial infection site. “Exopolysaccharidedegradation” is the ability of a virus to produce a protein or enzymecapable of degrading or dissociating high-molecular weight compoundssecreted by microorganisms into their environment to form the structuralintegrity of biofilms. EPS degrading activities can include but are notlimited to surfactants, glycosidases, and proteases. Their activitiescan be measured using standard biochemical assays known to those skilledin the art. Modulation of quorum sensing molecules can also lead tobiofilm dispersion. Quorum sensing molecules are known to be highlyconserved regulators of virulence in a number of human pathogenicbacteria. Proteins with enzymatic activities capable of degrading quorumsensing molecules have been identified and their activities measuredthrough various microbial reporter assays, biochemical reporter assays,or by analysis of cleavage products using TLC (Rajesh and Rai,Microbiological Research, July-August 2014, Volume 169, Issues 7-8,Pages 561-569, incorporated herein by reference). Degradation ofextracellular DNA or RNA within a biofilm or bacterial infection sitecan also lead to biofilm dispersion. Viral encoded DNase or RNaseactivities can be measured through commercially available kits known tothose skilled in the art, such as those available from Jena Bioscienceor Thermofisher as non-limiting examples. Biofilm prevention,penetration, destruction, or dispersion can also be assessed byquantifying the biofilm present after treatment and comparing it to acontrol condition. Biofilm measurements are well known in the art andinclude, as a non-limiting example, staining the biofilm with a dye,such as crystal violet, and quantifying the absorbance on aspectrophotometer.

In some examples, the present disclosure provides a method ofengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, bacterial phage resistance. Phage orbacteriophage are terms that can be used interchangeable and refer toviruses that infect bacteria. Bacterial phage resistance refers to theemergence of bacteriophage-resistant bacteria from a population treatedwith or exposed to a specific virus. This occurs either through randommutations within the bacteria, or because certain bacteria within thepopulation were not able to be infected by the virus. When theseresistant bacteria expand, the new population is resistant to the virusor bacteriophage it was originally exposed to. A non-limiting example ofassessing bacterial resistance is to track the rate of bacterial growthfollowing viral treatment, as the number of resistant bacteria directlyinfluence the speed of population re-growth. Bacteriophage can beengineered to prevent bacteria from acquiring viral resistance by atleast three methods, including 1) inhibiting known viral resistancesystems, 2) encoding a secondary toxin, and/or 3) increased virulencethrough increased lytic capacity. Bacteriophage can avoid or inhibitknown viral resistance systems through expression of known or syntheticinhibitory proteins, as one example. Activity of these inhibitoryproteins can be monitored through the classic double-layer plaquetitration method and/or analysis of the efficiency of plating. The viralresistance systems can include, but are not limited to, CRISPR-Cas andrestriction modification systems. Prevention of viral resistance canalso be achieved through expression of secondary toxins, such asbactericidal payloads. The activity of these secondary toxins isindependent of the natural lytic activity of the given virus and can bemeasured through growth/kill curve analysis. Additionally, oralternatively, the genetically encoded toxic protein can be purified andcharacterized using established biochemical and/or phenotypic assayscommonly used to characterize protein toxins and that are well known byone skilled in the art.

In some examples, the present disclosure provides a method ofengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, bacterial antibiotic sensitization.“Bacterial antibiotic sensitization” refers to the ability of a virus toexpress a genetically encoded payload to make infected or neighboringcells more sensitive to an antimicrobial agent. The payload can begenetically encoded on the virus or bacteriophage and then expressedwithin the host cell. The expressed payload can optionally be secretedby the host cell or released upon host cell lysis. Antibioticsensitization activity can be observed through synergy testing using,for example, the well-known microdilution checkerboard assay.

In some examples, the present disclosure provides a method ofengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, modulation of virulence factors.“Modulation of virulence factors” refers to a virus genetically encodingproteins or compounds capable of modulating the expression or activityof known virulence factors. Non-limiting examples of virulence factormodulators are transcription factors, antibodies, and immunity proteins.The expression or activity of virulence factors and virulence factormodulators can be observed, for example, in animal models, biochemicaltests, or reporter assays.

In some examples, the present disclosure provides a method ofengineering a viral nucleic acid that results in an improved viralproperty, such as, for example, targeted host genome digestion orediting. “Targeted host genome digestion or editing” refers to theability of a virus to genetically encode a sequence-specific nucleasecapable of targeted genome digestion at a given genetic locus, andoptionally editing through, for example, insertion of a repair DNAmolecule. The targeted digestion activity can be observed throughsequencing, viable counts, confirmation of new sequence integration,and/or other standard techniques known to those skilled in the art.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising an in vitro digestion step.In some examples, the digested viral nucleic acid is isolated andsequenced in lieu of being used in the in vitro or in vivo assemblyreaction. In some examples, the sequencing results from the viralnucleic acid fragment is used to determine the viral genome termini. Insome examples, the corrected viral genome sequences are used to plan anddesign further in vitro engineering approaches and steps.

In some aspects, the present disclosure provides for an in vitro methodof engineering a viral sequence comprising isolation of a viral nucleicacid. In some examples, the viral nucleic acid is a complete viralgenome. In some examples, the complete viral genome is isolated from aviral particle. In some examples, the viral nucleic acid is a subsectionof the viral genome. In some examples, the viral nucleic acid is asubsection of the viral genome comprised in a plasmid. In some examples,the plasmid comprising the viral genome subsection is isolated from ahost cell. In some examples, the viral genome subsection has been clonedinto a plasmid, transformed into a host cell, and isolated prior to invitro engineering. In some examples, the viral nucleic acid issynthesized de novo. De novo synthesis can include synthesizing oligosand assembling them in vitro or in vivo using standard methods known inthe art. In some examples, the viral nucleic acid is amplified prior todigestion, such as, for example, PCR-amplified.

In some aspects, the present disclosure provides for a kit forengineering a viral sequence comprising (a) an isolated non-thermostable5′ to 3′ exonuclease that lacks 3′ exonuclease activity, (b) a crowdingagent, (c) an isolated thermostable non-strand-displacing DNA polymerasewith 3′ exonuclease activity, or a mixture of said DNA polymerase with asecond DNA polymerase that lacks 3′ exonuclease activity, (d) anisolated thermostable ligase, (e) a mixture of dNTPs, (f) a suitablebuffer, and (g) purified recombinant RNA-guided nuclease. In someexamples, the RNA-guided nuclease is Cas9 or Cas9 derived enzyme. Insome examples, the kit further comprises custom-designed targeting RNAs.In some examples, the targeting RNAs are chimeric gRNAs or crRNA andtracrRNA. In some examples, the kit further comprises custom-designedsynthesized nucleic acid molecules to serve as the inserted DNA fragmentin the assembly reaction. In some examples, the kit further comprisescompetent host cells. In some examples, the kit further comprisesisolated viral nucleic acids.

In some aspects, the present disclosure provides for a system for invitro engineering of a viral nucleic acid comprising isolated viralnucleic acid, recombinant RNA-guided nuclease, at least one targetingRNA, and a DNA or RNA fragment that will be assembled into the isolatedviral nucleic acid at the site of digestion. In some examples, theisolated viral nucleic acid is a complete genome isolated from viralparticles. In some examples, the isolated viral nucleic acid is a viralgenome subsection that was subcloned into a plasmid and isolated from ahost cell. In some examples, the RNA-guided nuclease is Cas9 or aCas9-derived enzyme. In some examples, the targeting RNA is a crRNA andtracrRNA. In some examples, the targeting RNA is a chimeric guide RNA(gRNA). In some examples, there are two targeting RNAs or gRNAs. In someexamples, there are two sets of crRNA and tracrRNA.

In some aspects, the present disclosure provides an in vitro engineeredviral nucleic acid system comprising: isolated viral nucleic acid,recombinant RNA-guided nuclease, at least one targeting RNA, and anucleic acid fragment to be inserted into the isolated nucleic aciddigestion site. In some examples, the system is such that therecombinant RNA-guided nuclease and at least one targeting RNA form acomplex capable of digesting the isolated viral nucleic acid. In someexamples, the system further comprises spermidine. In some examples, thesystem further comprises: an isolated non-thermostable 5′ to 3′exonuclease that lacks 3′ exonuclease activity; a crowding agent; anisolated thermostable non-strand-displacing DNA polymerase with 3′exonuclease activity, or a mixture of said DNA polymerase with a secondDNA polymerase that lacks 3′ exonuclease activity; an isolatedthermostable ligase; a mixture of dNTPs; and a suitable buffer, whereinthe system is under conditions that are effective for insertion of thenucleic acid fragment into the isolated viral nucleic acid at the siteof RNA-guided nuclease digestion to form a recombinant viral nucleicacid.

In some aspects, the herein described system is such that therecombinant viral nucleic acid is capable of producing non-naturallyoccurring viral particles with at least one improved viral propertycompared to the reference and/or non-engineered viral nucleic acid. Insome examples, the improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing.

In some aspects, in the herein described system, the RNA-guided nucleaseis Cas9 or a Cas9-derived enzyme. In some examples, the RNAguided-nuclease is inactivated or removed following digestion.

The herein disclosed method can be used in multiple other viral genomesand viral vector constructs, used to modify RNA genomes by directlyediting the RNA genome or a DNA template that will then be in vitrotranscribed into the viral RNA, used to engineer and directly modifyboth Prokaryotic and Eukaryotic viruses, and used to directly modifyviral genomes used for phage display, phage therapy, viral diagnostics,or vaccine development/production.

In some aspects, the present disclosure provides a recombinant viralnucleic acid generated by any of the methods described herein. In someexamples, the recombinant viral nucleic acid is capable of producingnon-naturally occurring viral particles with at least one improved viralproperty compared to the non-engineered viral nucleic acid. In someexamples, the improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing.

In some aspects, the present disclosure provides an engineered viralcomposition comprising a recombinant nucleic acid capable of producingnon-naturally occurring viral particles with at least one improved viralproperty compared to the non-engineered viral nucleic acid. In someexamples, the improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing. Insome examples, the engineered viral nucleic acid according to thepresent disclosure is generated by any of the steps in the hereindescribed methods.

The method may be used to alter a nucleotide, gene, or whole genomicregion. For example, as described in the examples below, this method hasbeen shown to substitute the LKD16 gp18 gene into LUZ19 resulting inimproved viral host range. Additionally, this method may be used toinsert a single mutation in the viral tubular complex to improve viralreplication. The method may also be used to engineer antimicrobialpeptides; pyocins; EPS-depolymerases; CRISPR/Cas inhibitory proteins;tail fibers from bacteriophage; reporter genes (i.e. Lux, GFP);Quorum-quenching genes; nucleases; TALEN nucleases; Type I, Type II,Type III, Type IV, Type V, and Type VI CRISPR system proteins (i.e.Cas9); CRISPR RNAs, transcription factors and human immune modulatingfactors into a bacteriophage to improve activity of the bacteriophage inbacteriophage therapy or related uses. These elements can by operablylinked to a native or heterologous regulatory elements, such as a nativepromoter, heterologous promoter, inducible promoter, or any combinationthereof.

In some embodiments, the present disclosure provides an engineered viruscomprising an engineered viral nucleic acid capable, upon introductioninto a host cell, of producing non-naturally occurring viral particleswith two or more improved viral properties compared to thenon-engineered viral nucleic acid. In some aspects, the produced viralparticles have at least three improved viral properties. In someaspects, each improved viral property is selected from the groupconsisting of host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing.

In some embodiments, the present disclosure provides an engineered viruscomprising an engineered viral nucleic acid. In some aspects, theengineered viral nucleic acid is an engineered viral genome. In someaspects, the engineered viral genome is an engineered bacteriophagegenome. In some aspects of the engineered bacteriophage, at least one ofthe improved viral properties is host range.

In some embodiments, the present disclosure provides an engineeredvirus, with two or more improved viral properties, which comprises anengineered viral nucleic acid. In some aspects, each improved viralproperty is the result of at least one modification in the engineeredviral nucleic acid. In some aspects, at least one improved viralproperty is the result of at least two modifications in the engineeredviral nucleic acid. In some aspects, the modifications comprised in theengineered viral nucleic acid are the result of a single engineeringstep. In some aspects, the modifications comprised in the engineeredviral nucleic acid are the result of iterative engineering steps.

In some embodiments, the present disclosure provides an engineeredvirus, with two or more improved viral properties, which comprises anengineered viral nucleic acid.

In some aspects, at least one of the modifications is within a nucleicacid sequence having at least 50%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, 100% or complete identity to a sequence comprised within SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:50, or SEQ IDNO:25.

In some aspects, at least one of the modifications is within a nucleicacid sequence encoding an amino acid sequence having at least 50%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, 100% or complete identity to SEQID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:5, SEQ ID NO:48, or SEQID NO:49.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises all or a portionof a heterologous gp18 gene. In some aspects, the heterologous gp18 genehas at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% orcomplete identity to SEQ ID NO:26. In some aspects, the heterologousgp18 gene encodes an amino acid sequence with at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, 100% or complete identity to SEQ IDNO:38.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises all or a portionof an engineered gp34 gene. In some aspects, the engineered gp34 geneencodes an amino acid sequence comprising a mutation at a positioncorresponding to amino acid position 55 of SEQ ID NO:5. In some aspects,the heterologous gp34 gene has at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:4.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises a modification inone or more sequences having at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to a sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:50.

In some aspects, the engineered viral genome further comprises amodification in each of a sequence having at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to SEQ ID NO:1, asequence having at least 50%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,100% or complete identity to SEQ ID NO:2, a sequence having at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, 100% or complete identityto SEQ ID NO:3, and a sequence having at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to SEQ ID NO:50. Insome aspects, the modifications comprise a G to A replacement at aposition corresponding to nucleic acid position 50 of SEQ ID NO:1, a Gto T replacement at a position corresponding to nucleic acid position160 of SEQ ID NO:50, a A to G replacement at a position corresponding tonucleic acid position 245 of SEQ ID NO:2, a AT to TC replacement atpositions corresponding to nucleic acid positions 247-248 of SEQ IDNO:2, and a A to G replacement at a position corresponding to nucleicacid position 757 of SEQ ID NO:3.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises a modification inone or more nucleic acid sequences encoding an amino acid sequencehaving at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% orcomplete identity to a sequence selected from the group consisting ofSEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:48.

In some aspects, the engineered viral genome comprises a modification ina nucleic acid sequence encoding each of an amino acid sequence havingat least 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, 100% or completeidentity to SEQ ID NO:34, an amino acid sequence having at least 50%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, 100% or complete identity to SEQID NO:35, an amino acid sequence having at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to SEQ ID NO:36, andan amino acid sequence having at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:48. In some aspects,the modifications comprise a C to Y replacement at a positioncorresponding to amino acid position 17 of SEQ ID NO:34, a D to Yreplacement at a position corresponding to amino acid position 36 of SEQID NO:48, a D to G replacement at a position corresponding to amino acidposition 82 of SEQ ID NO:35, a I to S replacement at positioncorresponding to amino acid position 83 of SEQ ID NO:35, and a N to Dreplacement at a position corresponding to amino acid position 253 ofSEQ ID NO:36.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises a modificationwithin a sequence having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:25. In some aspects,the modification is an insertion of a heterologous nucleic acid moleculeinto a sequence having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:25, or a replacementof a sequence comprised within a sequence having at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, 100% or complete identity to SEQ IDNO:25 with a heterologous nucleic acid molecule. In some aspects, theheterologous nucleic acid molecule comprises a heterologous nucleic acidsequence having at least 50%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,100% or complete identity to a sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

In some aspects, the engineered viral genome comprises all or a portionof a viral genome having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to the LUZ19 genome. In someaspects, the engineered viral genome further comprises a modificationwithin a nucleic acid sequence encoding an amino acid sequence having atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, 100% or completeidentity to SEQ ID NO:49. In some aspects, the modification is aninsertion of a heterologous nucleic acid molecule into a nucleic acidsequence encoding an amino acid sequence having at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, 100% or complete identity to SEQ IDNO:49, or a replacement of a nucleic acid sequence comprised within anucleic acid sequence encoding an amino acid sequence having at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, 100% or complete identityto SEQ ID NO:49 with a heterologous nucleic acid molecule. In someaspects, the heterologous nucleic acid molecule comprises a heterologousnucleic acid sequence encoding an amino acid sequence having at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, 100% or complete identityto a sequence selected from the group consisting of SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, and SEQ ID NO:47.

In some aspects, the engineered viral nucleic acid comprises aheterologous nucleic acid sequence operably linked to a promotercomprising a nucleic acid sequence comprised within SEQ ID NO:21 or aportion thereof.

In some aspects, the engineered viral nucleic acid comprises aheterologous nucleic acid sequence operably linked to a terminatorcomprising a nucleic acid sequence comprised within SEQ ID NO:22 or aportion thereof.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties comprising: (a) providing a first viral genome; and (b)engineering a second viral genome by combining at least one fragment ofthe first viral genome with at least one repair nucleic acid moleculesuch that the resulting second viral genome comprises at least onemodification compared to the first viral genome, and wherein, upon beingintroduced into a host cell, the second viral genome is capable ofproducing viral particles with two or more improved viral properties. Insome aspects, the method disclosed herein further comprises (c)repeating steps (a)-(b) in one or more iterations. In some aspects, eachimproved viral property is selected from the group consisting of hostrange, viral lytic cycle, adsorption, attachment, injection, replicationand assembly, lysis, burst size, immune evasion, immune stimulation,immune deactivation, biofilm dispersion, bacterial phage resistance,bacterial antibiotic sensitization, modulation of virulence factors, andtargeted host genome digestion or editing.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, engineering thesecond viral genome in step (b) further comprises: (1) in vitrodigestion of a region of the first viral genome using an endonuclease;and (2) assembling at least one fragment of the digested first viralgenome with at least one repair nucleic acid molecule. In some aspects,the first viral genome is isolated from viral particles. In someaspects, the first viral genome or the at least one repair nucleic acidmolecule is synthesized de novo. In some aspects, de novo synthesiscomprises combining chemically synthesized nucleic acid molecules,PCR-amplified nucleic acid sequences, digested fragments of isolatednucleic acid molecules, or any combination thereof. In some aspects, thefirst viral genome or the at least one repair nucleic acid molecule isamplified prior to in vitro digestion.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the first viralgenome is at least 18 kb. In some aspects, the first viral genome isbetween at least 2 kb and at least 4 Mb. In some aspects, the firstviral genome is between at least 18 kb and at least 4 Mb. In someaspects, the first viral genome is at least 5 kb, at least 10 kb, atleast 15 kb, at least 18 kb, at least 20 kb, at least 25 kb, at least 30kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, atleast 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 100 kb, atleast 125 kb, at least 150 kb, at least 175 kb, at least 200 kb, atleast 250 kb, at least 300 kb, at least 400 kb, at least 500 kb, atleast 600 kb, at least 700 kb, at least 800 kb, at least 900 kb, atleast 1 Mb, at least 1.5 Mb, at least 2 Mb, at least 2.5 Mb, at least 3Mb, or at least 3.5 Mb.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the assembly isperformed in vitro or in vivo. In some aspects, the assembly isperformed in vitro with a mixture comprising: (a) an isolatednon-thermostable 5′ to 3′ exonuclease that lacks 3′ exonucleaseactivity; (b) a crowding agent; (c) an isolated thermostablenon-strand-displacing DNA polymerase with 3′ exonuclease activity, or amixture of said DNA polymerase with a second DNA polymerase that lacks3′ exonuclease activity; (d) an isolated thermostable ligase; (e) amixture of dNTPs; and (f) a suitable buffer, under conditions that areeffective for insertion of the fragment into the digested viral nucleicacid to form a recombinant nucleic acid comprising the engineered viralgenome.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the assembly isperformed in vitro or in vivo. In some aspects, the assembly isperformed in vivo in a host cell.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the endonucleaseis an RNA-guided nuclease. In some aspects, the method further comprisesone or two guiding RNAs. In some aspects, the RNA-guided nuclease isCas9 or a Cas9 derived enzyme. In some aspects, the guiding RNAscomprise 1) a chimeric gRNA or 2) a crRNA and tracrRNA.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the endonucleaseis heat inactivated or removed. In some aspects, the in vitro digestionfurther comprises spermidine.

In some embodiments, the present disclosure provides a method forgenerating an engineered virus of interest having two or more desiredviral properties as describe herein. In some aspects, the method furthercomprises transforming of the engineered viral genome into a host cell.In some aspects, the method further comprises using an in vitropackaging kit for packaging of the engineered viral genome into viralparticles.

In some embodiments, the present disclosure provides an engineered virusgenerated by any of the methods disclosed herein.

In some embodiments, the present disclosure provides compositions of anyof the engineered viruses disclosed herein generated by any of theengineering methods disclosed herein.

In some embodiments, the present disclosure provides a kit forengineering viral nucleic acid molecules comprising: purifiedrecombinant RNA-guided nuclease; an isolated non-thermostable 5′ to 3′exonuclease that lacks 3′ exonuclease activity; a crowding agent; anisolated thermostable non-strand-displacing DNA polymerase with 3′exonuclease activity, or a mixture of said DNA polymerase with a secondDNA polymerase that lacks 3′ exonuclease activity; an isolatedthermostable ligase; a mixture of dNTPs; and a suitable buffer. In someaspects, the kit further comprising custom-designed guide RNAs. In someaspects, the kit further comprising custom-designed synthesized nucleicacid molecules to serve as the inserted DNA fragment in an assemblyreaction. In some aspects, the kit further comprising competent hostcells for transformation. In some aspects, the kit further comprisingisolated viral genomic nucleic acids.

In some aspects, the present disclosure provides an in vitro engineeredviral nucleic acid system comprising: isolated viral nucleic acid,recombinant RNA-guided nuclease, at least one targeting RNA, and anucleic acid fragment to be inserted into the isolated nucleic aciddigestion site. In some examples, the system is such that therecombinant RNA-guided nuclease and at least one targeting RNA form acomplex capable of digesting the isolated viral nucleic acid. In someexamples, the system further comprises spermidine. In some examples, thesystem further comprises: an isolated non-thermostable 5′ to 3′exonuclease that lacks 3′ exonuclease activity; a crowding agent; anisolated thermostable non-strand-displacing DNA polymerase with 3′exonuclease activity, or a mixture of said DNA polymerase with a secondDNA polymerase that lacks 3′ exonuclease activity; an isolatedthermostable ligase; a mixture of dNTPs; and a suitable buffer, whereinthe system is under conditions that are effective for insertion of thenucleic acid fragment into the isolated viral nucleic acid at the siteof RNA-guided nuclease digestion to form a recombinant viral nucleicacid.

In some aspects, the herein described system is such that therecombinant viral nucleic acid is capable of producing non-naturallyoccurring viral particles with at least one improved viral propertycompared to the non-engineered viral nucleic acid. In some examples, theimproved viral property is selected from the group consisting of hostrange, viral lytic cycle, adsorption, attachment, injection, replicationand assembly, lysis, burst size, immune evasion, immune stimulation,immune deactivation, biofilm dispersion, bacterial phage resistance,bacterial antibiotic sensitization, modulation of virulence factors, andtargeted host genome digestion or editing.

In some aspects, in the herein described system, the RNA-guided nucleaseis Cas9 or a Cas9-derived enzyme. In some examples, the RNAguided-nuclease is inactivated or removed following digestion.

In some aspects, the herein described method is used as an errorcorrection method to correct sequences in isolated nucleic acids.Standard error correction methods are PCR-based, which has two inherentproblems: 1) PCR can introduce additional unwanted mutations into thenucleic acid and 2) PCR, in this context, has a size restriction ofapproximated 5 kb. Therefore, standard PCR-based error correctionmethods cannot reliably be performed on plasmids larger than 5 kb,either as a result of PCR-generated mutations or a failure to amplify.The herein described method of in vitro engineering of a nucleic acidsequence circumvents the need for PCR amplification, which removes thesize restriction and eliminates the possibility of PCR-generatedmutations.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising isolation of a nucleicacid; in vitro digestion of a region of the nucleic acid using aRNA-guided nuclease; and assembly of a recombinant nucleic acid by theinsertion of a DNA or RNA fragment into the digested nucleic acid. Inone aspect, the in vitro digestion is an RNA-guided enzymatic digestion.In another aspect, the enzymatic digestion is performed using Cas9 or aCas9 derived enzyme. In an additional aspect, the digestion furthercomprises targeting RNAs. In another aspect, the digestion furthercomprises spermidine. In a specific aspect, the targeting RNAs are gRNA,crRNA and/or tracrRNA. In a further aspect, following digestion, theRNA-guided nuclease is inactivated by standard methods such as exposureto heat, for example, such as at least 80° Celcius. Additionally oralternatively, the RNA-guided nuclease is removed by standard methods,such as, for example, phenol-chloroform extraction.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising isolation of a nucleicacid; in vitro digestion of a region of the nucleic acid using aRNA-guided nuclease; and assembly of a recombinant nucleic acid by theinsertion of a DNA or RNA fragment into the digested nucleic acid. Insome examples, the assembly is performed in vitro in a single vesselwith a mixture of components comprising (a) an isolated non-thermostable5′ to 3′ exonuclease that lacks 3′ exonuclease activity, (b) a crowdingagent, (c) an isolated thermostable non-strand-displacing DNA polymerasewith 3′ exonuclease activity, or a mixture of said DNA polymerase with asecond DNA polymerase that lacks 3′ exonuclease activity, (d) anisolated thermostable ligase, (e) a mixture of dNTPs, and (f) a suitablebuffer, under conditions that are effective for insertion of thefragment into the digested viral nucleic acid to form a recombinantnucleic acid. In some aspects, the exonuclease is a T5 exonuclease andthe contacting is under isothermal conditions, and/or the crowding agentis PEG, and/or the non-strand-displacing DNA polymerase is Phusion™ DNApolymerase or VENT® DNA polymerase, and/or the ligase is Taq ligase. Insome examples, the in vitro assembly is performed by one-step orisothermal Gibson assembly. In some examples, the in vitro assembly isperformed by two-step Gibson assembly.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising an RNA-guidednuclease. In some examples, the RNA-guided nuclease is a Type II Cas9.In some examples, the RNA-guided nuclease is Cas9 or a Cas9 derivedenzyme. In some examples, the RNA-guided nuclease is an isolatedrecombinant Cas9 or Cas9 derived enzyme. In some examples, there is atleast one targeting RNA. In some examples, there are two targeting RNAs.In some examples, the targeting RNA is a chimeric guide RNA (gRNA) or aset of a crRNA and tracrRNA. In some examples, the in vitro digestionreaction uses two gRNAs. In some examples, the in vitro digestionreaction uses two sets of crRNAs and tracrRNAs.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising an in vitro digestionstep. In some examples, following digestion, the RNA-guided nuclease isinactivated by standard methods such as exposure to heat, for example,such as at least 80° Celcius. In some examples, following digestion, theRNA-guided nuclease is removed by phenol-chloroform extraction. In someexamples, following digestion, the RNA-guided nuclease is removed byother extraction methods well known in the art.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence resulting in an engineerednucleic acid. In some examples, the engineered nucleic acid is thentransformed into a host cell. In some examples, the host cell is E.coli, P. aeruginosa, S. cerevisiae, V. natriegens, B. subtilis, or othermicroorganism well known in the art. In some examples, thetransformation is performed by heat shock, electroporation, biolistics,particle bombardment, conjugation, transduction, lipofection, or otherestablished method well known in the art.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising an isolated nucleicacid. In some examples, the nucleic acid is a complete genome isolatedfrom a host cell. In some examples, the host cell is E. coli, S.cerevisiae, B. subtilis, V. natriegens, P. aeruginosa or otherwell-known microorganism. In some examples, the nucleic acid is aplasmid. In some examples, the plasmid is isolated from a host cell. Insome examples, nucleic acid of interest has been cloned into a plasmid,transformed into a host cell, and isolated prior to in vitro engineeringvia the herein described method.

In some aspects, the present disclosure provides for an in vitro methodof engineering a nucleic acid sequence comprising isolation of a nucleicacid. In some examples, the isolated nucleic acid is a genome orplasmid. In some examples, the isolated genome or plasmid is at least 6kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, atleast 12 kb, at least 15 kb, at least 20 kb, at least 25 kb, or at least28 kb. In some examples, the isolated genome or plasmid is between 6 kband 1 MB. In some examples, the isolated genome or plasmid is between: 6kb and 10 kb, 8 kb and 15 kb, 12 kb and 20 kb, 15 kb and 22 kb, 20 kband 25 kb, 22 kb and 28 kb, 25 kb and 30 kb, 25 kb and 50 kb, or 40 kbto 100 kb.

Additionally or alternatively, to any of the above-disclosedembodiments, the disclosure comprises the following embodiments:

Embodiment 1 is an engineered virus comprising an engineered viralnucleic acid capable, upon introduction into a host cell, of producingnon-naturally occurring viral particles with two or more, or optionallythree or more, improved viral properties compared to the viral particlesproduced by introduction of the non-engineered viral nucleic acid into ahost cell.

Embodiment 2 is the engineered virus of Embodiment 1, wherein eachimproved viral property is selected from the group consisting of hostrange, viral lytic cycle, adsorption, attachment, injection, replicationand assembly, lysis, burst size, immune evasion, immune stimulation,immune deactivation, biofilm dispersion, bacterial phage resistance,bacterial antibiotic sensitization, modulation of virulence factors, andtargeted host genome digestion or editing.

Embodiment 3 is the engineered virus of Embodiment 1 or 2, wherein theviral nucleic acid is one or more of the following viral nucleic acids:viral genome, viral genome fragment, bacteriophage genome, bacteriophagegenome fragment, lytic bacteriophage genome, lytic bacteriophage genomefragment, or any combination thereof.

Embodiment 4 is the engineered virus of any of Embodiments 1-3, whereinthe engineered viral nucleic acid is a bacteriophage genome, andoptionally wherein at least one of the improved viral properties is hostrange.

Embodiment 5 is the engineered virus of any of Embodiments 1-4, whereinat least one of the following is satisfied: 1) each improved viralproperty is the result of at least one modification in the engineeredviral nucleic acid, 2) at least one improved viral property is theresult of at least two modifications in the engineered viral nucleicacid, 3) the modifications comprised in the engineered viral nucleicacid are the result of a single engineering step, 4) the modificationscomprised in the engineered viral nucleic acid are the result ofiterative engineering steps, or 5) any combination thereof.

Embodiment 6 is the engineered virus of any of Embodiments 1-5, whereinat least one of the modifications is within:

1) a nucleic acid sequence having at least 50%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, 100% or complete identity to a sequence comprisedwithin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:50,or SEQ ID NO:25, or

2) a nucleic acid sequence encoding an amino acid sequence having atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, 100% or completeidentity to SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:5, SEQID NO:48, or SEQ ID NO:49, or

3) any combination thereof.

Embodiment 7 is the engineered virus of any of Embodiments 1-6, whereinthe engineered viral nucleic acid comprises an engineered viral genomecomprising all or a portion of a viral genome having at least 50%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, 100% or complete identity to theLUZ19 genome.

Embodiment 8 is the engineered virus of any of Embodiments 1-7, whereinthe engineered viral genome further comprises at least one of thefollowing:

1) all or a portion of a heterologous gp18 gene, and optionally whereinthe heterologous gp18 gene has at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:26;

2) all or a portion of a heterologous gp18 gene, and optionally whereinthe heterologous gp18 gene encodes an amino acid sequence with at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, 100% or complete identityto SEQ ID NO:38;

3) all or a portion of an engineered gp34 gene, and optionally where theheterologous gp34 gene encodes an amino acid sequence comprising amutation at a position corresponding to amino acid position 55 of SEQ IDNO:5, or optionally wherein, the heterologous gp34 gene has at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, 100% or complete identityto SEQ ID NO:4;

4) a modification in one or more sequences having at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, 100% or complete identity to a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, and SEQ ID NO:50,

and optionally a modification in each of a sequence having at least 50%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, 100% or complete identity to SEQID NO:1, a sequence having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:2, a sequence havingat least 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, 100% or completeidentity to SEQ ID NO:3, and a sequence having at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, 100% or complete identity to SEQ IDNO:50,

and optionally wherein the modifications comprise a G to A replacementat a position corresponding to nucleic acid position 50 of SEQ ID NO:1,a G to T replacement at a position corresponding to nucleic acidposition 160 of SEQ ID NO:50, a A to G replacement at a positioncorresponding to nucleic acid position 245 of SEQ ID NO:2, a AT to TCreplacement at positions corresponding to nucleic acid positions 247-248of SEQ ID NO:2, and a A to G replacement at a position corresponding tonucleic acid position 757 of SEQ ID NO:3;

5) a modification in one or more nucleic acid sequences encoding anamino acid sequence having at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to a sequence selected from thegroup consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ IDNO:48,

and optionally a modification in a nucleic acid sequence encoding eachof an amino acid sequence having at least 50%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, 100% or complete identity to SEQ ID NO:34, an aminoacid sequence having at least 50%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, 100% or complete identity to SEQ ID NO:35, an amino acid sequencehaving at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% orcomplete identity to SEQ ID NO:36, and an amino acid sequence having atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, 100% or completeidentity to SEQ ID NO:48,

and optionally, wherein the modifications comprise a C to Y replacementat a position corresponding to amino acid position 17 of SEQ ID NO:34, aD to Y replacement at a position corresponding to amino acid position 36of SEQ ID NO:48, a D to G replacement at a position corresponding toamino acid position 82 of SEQ ID NO:35, a I to S replacement at positioncorresponding to amino acid position 83 of SEQ ID NO:35, and a N to Dreplacement at a position corresponding to amino acid position 253 ofSEQ ID NO:36;

6) a modification within a sequence having at least 50%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to SEQ ID NO:25,

and optionally wherein the modification is an insertion of aheterologous nucleic acid molecule into a sequence having at least 50%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, 100% or complete identity to SEQID NO:25, or a replacement of a sequence comprised within a sequencehaving at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% orcomplete identity to SEQ ID NO:25 with a heterologous nucleic acidmolecule,

and optionally wherein the heterologous nucleic acid molecule comprisesa heterologous nucleic acid sequence having at least 50%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to a sequenceselected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, and SEQ ID NO:20;

7) a modification within a nucleic acid sequence encoding an amino acidsequence having at least 50%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,100% or complete identity to SEQ ID NO:49,

and optionally wherein the the modification is an insertion of aheterologous nucleic acid molecule into a nucleic acid sequence encodingan amino acid sequence having at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 100% or complete identity to SEQ ID NO:49, or a replacementof a nucleic acid sequence comprised within a nucleic acid sequenceencoding an amino acid sequence having at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, 100% or complete identity to SEQ ID NO:49 witha heterologous nucleic acid molecule,

and optionally wherein the heterologous nucleic acid molecule comprisesa heterologous nucleic acid sequence encoding an amino acid sequencehaving at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% orcomplete identity to a sequence selected from the group consisting ofSEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47,

8) any combination thereof.

Embodiment 9 is the engineered virus of any of Embodiments 1-8, whereinthe engineered viral nucleic acid comprises a heterologous nucleic acidsequence operably linked to 1) a promoter comprising a nucleic acidsequence comprised within SEQ ID NO:21 or a portion thereof, 2) aterminator comprising a nucleic acid sequence comprised within SEQ IDNO:22 or a portion thereof, or 3) any combination thereof.

Embodiment 10 is a method for generating an engineered virus of interesthaving two or more desired viral properties comprising: (a) providing afirst viral genome; and (b) generating an engineered viral genome bycombining at least one fragment of the first viral genome with at leastone repair nucleic acid molecule to generate a second viral genomecomprising at least one modification compared to the first viral genome;wherein, the second viral genome, upon being introduced into a hostcell, is capable of producing viral particles with two or more improvedviral properties, and optionally (c) repeating steps (a)-(b) in one ormore iterations.

Embodiment 11 is the method of Embodiment 10, wherein each improvedviral property is selected from the group consisting of host range,viral lytic cycle, adsorption, attachment, injection, replication andassembly, lysis, burst size, immune evasion, immune stimulation, immunedeactivation, biofilm dispersion, bacterial phage resistance, bacterialantibiotic sensitization, modulation of virulence factors, and targetedhost genome digestion or editing.

Embodiment 12 is the method of either Embodiment 10 or 11, whereingenerating an engineered viral genome in step (b) comprises: (1) invitro digestion of a region of the first viral genome using anendonuclease; and (2) assembling at least one fragment of the digestedfirst viral genome with at least one repair nucleic acid molecule.

Embodiment 13 is the method of any of Embodiments 10-12, wherein atleast one of the following elements is satisfied: 1) the first viralgenome is isolated from viral particles, 2) the first viral and/or theat least one repair nucleic acid molecule is synthesized de novo, andoptionally wherein de novo synthesis comprises combining chemicallysynthesized nucleic acid molecules, PCR-amplified nucleic acidsequences, digested fragments of isolated nucleic acid molecules, or anycombination thereof, 3) the first viral genome and/or the at least onerepair nucleic acid molecule is amplified prior to in vitro digestions,or 4) any combination thereof.

Embodiment 14 is the method of any of Embodiments 10-13, wherein thefirst viral genome is at least one of the following:

1) at least 3 kb, at least 10 kb, at least 18 kb, at least 25 kb, or atleast 30 kb;

2) at least 18 kb;

3) between at least 2 kb and at least 4 Mb;

4) between at least 18 kb and at least 4 Mb; or

5) at least 5 kb, at least 10 kb, at least 15 kb, at least 18 kb, atleast 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, atleast 65 kb, at least 70 kb, at least 75 kb, at least 80 kb, at least 85kb, at least 90 kb, at least 100 kb, at least 125 kb, at least 150 kb,at least 175 kb, at least 200 kb, at least 250 kb, at least 300 kb, atleast 400 kb, at least 500 kb, at least 600 kb, at least 700 kb, atleast 800 kb, at least 900 kb, at least 1 Mb, at least 1.5 Mb, at least2 Mb, at least 2.5 Mb, at least 3 Mb, or at least 3.5 Mb.

Embodiment 15 is the method of any of Embodiments 10-14, wherein theassembly is performed in vitro, and optionally wherein the assembly isperformed in vitro with a mixture comprising: (a) an isolated 5′ to 3′exonuclease that lacks 3′ exonuclease activity which is optionallynon-thermostable; (b) optionally a crowding agent; (c) an isolatednon-strand-displacing DNA polymerase with 3′ exonuclease activity whichis optionally thermostable, or a mixture of said DNA polymerase with asecond DNA polymerase that lacks 3′ exonuclease activity; (d) anisolated ligase which is optionally thermostable; (e) a mixture ofdNTPs; and (f) optionally a suitable buffer, under conditions that areeffective for insertion of the fragment into the digested viral nucleicacid to form a recombinant nucleic acid comprising the engineered viralgenome.

Embodiment 16 is the method of any of Embodiments 10-14, wherein theassembly is performed in vivo, and optionally wherein the in vivoassembly is performed in a host cell.

Embodiment 17 is the method of any of Embodiments 10-16, wherein atleast one of the following elements is satisfied: 1) the endonuclease isan RNA-guided nuclease, 2) the method further comprises at least oneguiding RNA, 3) the RNA-guided nuclease is Cas9 or a Cas9-derived enzymeand wherein the at least one guiding RNA comprises (a) a chimeric gRNAor (b) a crRNA and tracrRNA, 4) the endonuclease is heat inactivated orremoved prior to assembly, 5) the in vitro digestion further comprisesspermidine, 6) the method further comprises transforming the engineeredviral genome into a host cell, 7) the method further comprises using anin vitro packaging kit for packaging of the engineered viral genome intoviral particles, or 8) any combination thereof.

Embodiment 18 is an engineered virus generated by the method of any ofthe Embodiments 10-17, and optionally wherein the engineered virus isthe engineered viruses from any of Embodiments 1-9.

Embodiment 19 is a kit for engineering nucleic acid molecules, which areoptionally viral nucleic acid molecules, comprising: (a) purifiedrecombinant RNA-guided nuclease; (b) an isolated 5′ to 3′ exonucleasethat lacks 3′ exonuclease activity which is optionally non-thermostable;(c) an isolated non-strand-displacing DNA polymerase with 3′ exonucleaseactivity which is optionally thermostable, or a mixture of said DNApolymerase with a second DNA polymerase that lacks 3′ exonucleaseactivity; (d) an isolated ligase which is optionally thermostable; andoptionally further comprising any of the following: 1) a crowding agent,2) a mixture of dNTPs, 3) a suitable buffer, 4) custom-designed guidingRNAs, 5) custom-designed synthesized nucleic acid molecules to serve asthe inserted DNA fragment in an assembly reaction, 6) competent hostcells for transformation, 7) isolated viral genomic nucleic acid, or 8)any combination thereof.

Embodiment 20 is a method of engineering a nucleic acid sequencecomprising: (a) providing a nucleic acid; (b) in vitro digestion of aregion of the nucleic acid using an RNA-guided nuclease; and (c)assembly of a recombinant nucleic acid by the insertion of a DNAfragment into the digested nucleic acid, wherein the assembly isperformed in vitro in a single vessel with a mixture of componentscomprising: (i) an isolated 5′ to 3′ exonuclease that lacks 3′exonuclease activity which is optionally non-thermostable; (ii) anisolated non-strand-displacing DNA polymerase with 3′ exonucleaseactivity which is optionally thermostable, or a mixture of said DNApolymerase with a second DNA polymerase that lacks 3′ exonucleaseactivity; (iii) an isolated ligase which is optionally thermostable;(iv) a mixture of dNTPs, under conditions that are effective forinsertion of the fragment into the digested nucleic acid to form arecombinant nucleic acid, and optionally wherein the in vitro assemblymixture further comprises (v) a crowding agent, or (vi) a suitablebuffer.

Embodiment 21 is the method of Embodiment 20, wherein at least one ofthe following elements is satisfied: 1) the RNA-guided nuclease is Cas9or a Cas9-derived enzyme, 2) the RNA-guided nuclease is heat inactivatedor removed prior to assembly, 3) the method further comprisestransformation of the recombinant nucleic acid into a host cell, 4) thenucleic acid is a plasmid isolated from a host cell, and optionallywherein the plasmid is at least 6 kb, at least 10 kb, at least 15 kb, orat least 20 kb, or 5) any combination thereof.

The disclosure in all its aspects is illustrated further in thefollowing Examples. The Examples do not, however, limit the scope of thedisclosure, which is defined by the appended claims. The discussion ofthe general methods given herein is intended for illustrative purposesonly. Other alternative methods and embodiments will be apparent tothose of skill in the art upon review of this disclosure, and are to beincluded within the spirit and purview of this application.

EXAMPLES Example I In Vitro Viral Genome Engineering

The 43 kb dsDNA LUZ19 viral genome (Accession number NC_010326.1) wasisolated from viral particles, for example using the Norgen Biotek phageDNA isolation kit or any other methods known to those in the art (FIG.2A). Site-specific digestion was performed using the RNA-guided nucleaseCas9 and in vitro transcribed gRNAs at two independent locations.Undigested 43 kb genomic DNA migrates considerably less than the largestDNA ladder band (10 kb). Digestion of linear genome yields fragments ofthree sizes: ˜39 kb, ˜4.3 kb, and ˜200 bp. Targeting gRNAs were used inexcess and obstruct the 200 bp fragment (FIG. 2B). A fragment of gp7from ΦKF77 was PCR amplified (FIG. 2C) using primers harboring 5′ tailswith 100 bp homology to regions directly upstream and downstream ofLUZ19 digestion sites. The Gibson Assembly method was used to integratethe PCR amplified ΦKF77 gp7 fragment (SEQ ID NO:8) seamlessly into thedigested LUZ19 genome to replace the native gp7 region (SEQ ID NO:23)(FIG. 2D). Little background is observed because Cas9 cleavage resultsin a blunt ended double stranded breaks which lack the homology requiredfor in vitro Gibson Assembly. The in vitro edited genomes weretransformed directly into host cells to yield functional viral particles(FIG. 2E). Integration of the ΦKF77 gene fragment into recovered viruseswas verified using PCR with primers internal and external to the regionof engineering. Unedited LUZ19 gDNA was used as a negative control,while all experimental viruses contained the new ΦKF77 gene fragment(last 7 lanes).

These data present an example of implementing in vitro viral engineeringto edit a P. aeruginosa lytic phage genome. Engineering phage such asLUZ19 cannot be done by standard methods due to toxicity effects inheterologous bacterial hosts such as E. coli, a lack of selectablemarkers appropriate for virulent viruses, and a lack of unique standardrestriction enzyme sites within the LUZ19 genome. Therefore, these datademonstrate how the herein described in vitro engineering method enablesdirect and rapid engineering of otherwise non-genetically tractableviral genomes.

For transformations into P. aeruginosa, chemically competent P.aeruginosa cells were prepared as described in Irani and Rowe (Irani, V.R. & Rowe, J. J. BioTechniques 1997, 22, 54-56). Basically, a 3 mlstarter culture of P. aeruginosa cells was diluted in 400 ml of freshLB. The culture was grown at 37° C. under shaking (220 rpm) to anOD₆₀₀=0.6 unless otherwise mentioned. Cells were chilled for 10 min onice, transferred into a 500 ml centrifuge bottle and pelleted in arefrigerated centrifuge (4° C.) at 5,000 g for 20 min. The bacterialpellet was washed with 100 ml of ice cold 150 mM MgCl₂ before beingsplit into two 50 ml conical tubes and pelleted at 5,000 g in arefrigerated centrifuge (4° C.). Cells were washed one additional timewith 30 ml 150 mM MgCl₂ before being centrifuged and resuspended in 15ml cold 150 mM MgCl₂. The cell suspension was incubated on ice for 1 hbefore being centrifuged at 4° C. and resuspended in 4 ml chilled 150 mMMgCl₂. Aliquots of 200 μl were placed into individual 1.5 mlmicrocentrifuge tubes and kept on ice for up to 2 days. Purified DNA wasadded to each aliquot of cells, briefly vortexed, and incubated on icefor an additional 1 h. Cells were heat shocked at 50° C. for 3 min andplaced directly back onto ice for 5 min before plating. Eachtransformation was added to 4 ml of 50° C. LB top agar and plated onto apre-warmed LB plate. Plates were inverted and incubated at 37° C. ON toallow plaque formation.

Example II Engineered Virus with Expanded Host Range

A large clinical library (282 P. aeruginosa isolates) was screened forsusceptibility to the phages LUZ19 and LKD16, using double agar plaqueassay. Sixty-six strains were able to be infected by at least one of thetwo viruses, with 18 and 6 strains being uniquely infected by LUZ19 andLKD16, respectively. Thus, LUZ19 was selected as a chassis for testingLKD16 genetic elements responsible for host range expansion. Comparativegenomics between the two viruses indicated that LKD16 gene product 18(gp18) had a distinct sequence from the LUZ19 gp18 homolog, indicatingit may be responsible for host range determination. The viral genome wasisolated from LUZ19 viral particles as described above. Site-specificdigestion was performed using an RNA-dependent nuclease and in vitrotranscribed gRNAs to excise the LUZ19 gp18 gene. The gp18 from LKD16 wasPCR amplified with LUZ19 homologous ends for integration. The GibsonAssembly method was used to integrate the PCR amplified LKD16 gp18 (SEQID NO:7) seamlessly into the digested LUZ19 genome in order to replacethe native gp18 sequence (SEQ ID NO:50). The in vitro engineered genomeswere transformed directly into host cells to yield functional viralparticles. The engineered LUZ19 virus harboring LKD16 gene gp18 was ableto infect all strains normally infected by the LUZ19 phage as well as 3strains previously infected only by LKD16, demonstrating host rangeexpansion (FIGS. 3B and 6B). This demonstrates that gp18 is a geneticelement responsible for differential LKD16 host range and that theengineered LUZ19 virus, harboring this gene, is better able to replicatein more host strains.

These data demonstrate implementing the herein disclosed in vitroengineering method to an otherwise non-genetically tractable viralgenome, which resulted in the improved viral property of expanded hostrange. The ability to rationally engineer bacteriophage with an expandedhost range is a property of great value when developing viruses to killbacteria.

Example III Engineered Virus with Host Range of a Viral Genus

LUZ19 and/or a LUZ19 derivative was used as starting material forevolution or co-infection experiments to identify targets for collapsingthe host range of the ΦKMV viral genus into a single representativevirus. Co-transformation or co-infection experiments were performedeither in a permissive (PAO1K) or a non-permissive (resistant) host(PA7410 or PA7649) (FIG. 4A). Both co-infection and co-transformationwere performed in the presence of LKD16 or ΦKMV, respectively. Hostrange was tested using the double agar plaque assay on indicatedbacterial strains. Following screening for expanded host range in thestrain of interest, the evolved phage was passaged 3-5 timesalternatively through permissive and selective strains (a strain that isinfected only by LUZ19-PA7632). Evolved phage were amplified in PAO1K,gDNA was purified and sequenced. Comparative genomics between LUZ19 andevolved LUZ19 capable of infecting strains previously sensitive only toLKD16 or ΦKMV was used to identify the point mutation responsible forhost range expansion (FIG. 4B).

A large clinical library (282 P. aeruginosa isolates) was screened forsusceptibility to ΦKMV genus of viruses, using the the double agarplaque assay. Three phage (LUZ19, LKD16, and ΦKMV) displayeddifferential host range and were able to infect 67 strains, with LUZ19infecting the majority of clinical isolates (FIG. 4C). Six clinicalisolates (PA7245, PA7255, PA7427, PA7503, PA7686, and PA7410) weresusceptible only to LKD16 and one clinical isolate was susceptible onlyto ΦKMV (PA7649). Thus, LUZ19 was selected as a chassis forevolution/co-infection/co-transformation experiments to obtain a variantable to infect all the clinical isolates sensitive to the ΦKMV genus.Comparative genomics revealed several point mutations were necessary forLUZ19 to infect strains susceptible only to LKD16 or ΦKMV: (i) gp13 C17Y(position 50 of SEQ ID NO:1) is necessary for infection of PA7427; (ii)gp18 D36Y (position 106 of SEQ ID NO:50) required for infection ofPA7245, PA7503 and PA7686; gp38 D82G and I83S (positions 245 and 247-248of SEQ ID NO:2 respectively) enables infection of PA7410 and PA7649;(iv) gp40 N253D (position 757 of SEQ ID NO:3) allows infection of PA7255(FIG. 4B). Iterative engineering of the above-mentioned mutations intoLUZ19 chassis using the herein described in vitro engineering methodresulted in a wide host range LUZ19 (WHR LUZ19) capable of infecting allthe clinical isolates susceptible to ΦKMV genus phage (FIG. 4C).

These data provide an example of using the herein disclosed in vitroengineering method to collapse the host range of a viral genus into asingle viral genome by first identifying the genetic mutationsresponsible for host range differences following evolution experiments,screening, sequencing, comparative genomics, and any combinationthereof.

Example IV

Improved Viral Replication Improves Early Biofilm Disruption

In another example, viral evolution and comparative genomics indicatedthat a LUZ19 evolved phage with a L55Δ mutation within the tail tubularprotein B (Gp34) replicated at a greater rate due to an increased burstsize (FIG. 5B). To validate that a Gp34 L55Δ mutation had improved viralproperties, the LUZ19 viral genome was isolated from viral particles.Site-specific digestion was performed using an RNA-dependent nucleaseand in vitro transcribed gRNAs to remove the gp34 gene (SEQ ID NO:4).The gp34 L55Δ gene, which harbors a deletion of leucine codon at aminoacid position 55 (Gp34 L55Δ, position 163-165 of SEQ ID NO:4) was PCRamplified from a LUZ19 evolved phage. The Gibson Assembly method wasused to integrate the PCR amplified gp34 L55Δ gene seamlessly into thedigested LUZ19 genome. The in vitro transformed genomes were transformeddirectly into host cells to yield functional viral particles. Theengineered LUZ19 virus harboring Gp34 L55Δ was able to diffuse and lysebacteria. Double agar plaque assays were used to show that LUZ19 phageharboring the gp34 L55Δ mutation (Phage*) had a larger zone of clearingthan wild type LUZ19. Images were taken and zones of clearing weremeasured over a two-day period (FIG. 5B). The expanding zone of lysiswidth indicates that viruses harboring Gp34 L55Δ mutations are betterable to diffuse and lyse bacteria. Crystal violet biofilm assay measuresbiofilm accumulation as a measure of the incorporation of crystal violet(FIG. 5C). Samples treated with viruses harboring gp34 L55Δ mutationshad a significant reduction in biofilm as compared to viruses with awild type gp34 gene. Illustration showing the location of the gp34mutation (asterisk) as compared to the wild type LUZ19 genome (FIG. 5D).Standard assays known in the art were used to measure viral adsorption,latent period, and burst size for both wild type and gp34 L55Δ mutants.These data indicated that viruses harboring a gp34 L55Δ mutation had agreatly increased burst size (FIG. 5E).

These data provide an example of using the herein disclosed in vitroengineering method to create a virus with the improved viral propertiesof increased bacterial lysis, burst size, replication, and early biofilmdisruption.

Example V Iterative Engineering Virus with Early Biofilm Disruption andExpanded Host Range

The expanded host range LUZ19_(LKD16gp18) recombinant viral genomecreated in Example II was isolated from viral particles. Site-specificdigestion was performed to remove gp34 (SEQ ID NO:4) using anRNA-dependent nuclease and in vitro transcribed gRNAs. The lyticactivity increasing gp34 ΔLeu55 mutation (position 163-165 of SEQ IDNO:4) characterized in Example IV was then PCR amplified and assembledinto the digested LUZ19_(LKD16gp18) viral genome using Gibson Assembly.The iteratively in vitro engineered genomes were transformed directlyinto host cells to yield functional viral particles, i.e. the engineeredLUZ19 virus harboring both the LKD16 gene gp18 and gp34 ΔLeu55 mutation(LUZ19*_(LKD16gp18)).

The LUZ19*_(LKD16gp18) virus was analyzed for improved viral properties,using double agar plaque, biofilm, and an in vitro human keratinocyteattachment assays. FIG. 6D demonstrates that LUZ19*_(LKD16gp18) hadimproved host range. LUZ19*_(LKD16gp18) was compared with native LUZ19for the ability to disrupt preformed MDR P. aeruginosa biofilms.Specifically, LUZ19*_(LKD16gp18) and wild type LUZ19 were incubated witha P. aeruginosa biofilm and disruption was measured using crystalviolet. FIG. 6E demonstrates that LUZ19^(*) _(LKD16gp18) has an enhancedability to disrupt preformed MDR P. aeruginosa biofilms compared withwild type LUZ19. The LUZ19*_(LKD16gp18) virus was analyzed for efficacyof phage treatment against bacteria attached to human keratinocytes.Specifically, P. aeruginosa were attached to a monolayer of HaCaT cells.The cells were then incubated with LUZ19*_(LKD16gp18) or wild typeLUZ19. The results indicated that the LUZ19*_(LKD16gp18) phage wasbetter able to kill multi-drug resistant (MDR)P. aeruginosa cellsattached to human keratinocytes (see FIG. 6F).

These data provide an example of how the herein described in vitroengineering method was used in a system to iteratively engineerbacteriophage with multiple independent improved viral properties, suchas expanded host range and increased burst size. Importantly, theseengineering steps would not be able to be performed as directly or atall using standard methods. Additionally, these data demonstrate theherein disclosed in vitro engineering method was used sequentially foriterative rounds of engineering, an important property for syntheticbiology applications.

Example VI Iterative Engineering Viruses with Biofilm-DispersingPayloads and Expanded Host Range Covering a Full Viral Genus

Either exopolysaccharide (EPS) depolymerases or phenol soluble modulins(PSM) were cloned into LUZ19 by replacing gp49 (SEQ ID NO:25), using theherein disclosed in vitro engineering method, to determine their abilityto disperse mature biofilm (FIG. 7). In order to engineer LUZ19 and WHRLUZ19 to express extracellular matrix depolymerase or surfactantpolypeptides, gp49 (SEQ ID NO:25) of LUZ19 or WHR LUZ19 was removed bydigestion using an RNA-dependent nuclease, in this case Cas9, and invitro transcribed gRNAs and subsequently replaced with the gene ofinterest (GOI) flanked by the major capsid promoter P_(gp32) (SEQ IDNO:21) and terminator T_(gp32) (SEQ ID NO:22) using Gibson Assembly(FIGS. 7A and 7C). In the case of wild type LUZ19, the GOI were EPSdepolymerases (Pp 15gp44-tail spike gp44 from Pseudomonas pudita Φ15(SEQ ID NO:14); NTUgp34-tail spike gp34 from Klebsiella pneumoniae phageNTU (SEQ ID NO:13); LKAlgp49-tail spike gp49 from P. aeruginosa phageLKA1 (SEQ ID NO:12)), surfactant phenol soluble modulins fromStaphylococcus epidermidis (PSMa-SEQ ID NO:18) and Staphylococcus aureus(PSMa3 (SEQ ID NO:16) and PSMb2 (SEQ ID NO:17)), and DspB surfactin fromAggregatibacter actinomycetemcomitans (SEQ ID NO:15) (FIG. 7B). In thecase of WHR LUZ19, the GOI were the EPS depolymerase Pp15gp44 (SEQ IDNO:14) and surfactin SePSMa (SEQ ID NO:18) (FIG. 7D). Engineered phagewere amplified within their appropriate host cell, isolated, andverified by sequencing.

Engineered phage ability to disperse mature biofilm was tested against a24 h biofilm grown in a MBEC device using 100 phage per well for 3 h.Briefly, overnight cultures of P. aeruginosa were diluted (1:100) in M63minimal medium supplemented with magnesium sulfate (1 mM), glucose(0.2%), and casamino acids (0.5%), and then added to sterile microtitreplates (150 μl per well). The lid with pegs was inserted in themicrotiter plate. After 24 h incubation at 37° C., the lid with pegs wasmoved to a microtiter plate containing 160 μl of complete MG63containing 100 phage per well. After 3 h incubation at 37° C., the lidwith pegs was washed 3 times in water, dried and stained with 200 μl of0.5% crystal violet. Subsequently, the plates were rinsed with water toremove unbound crystal violet and dried. The dye was dissolved in 200 μlof 30% acetic acid and the absorbance was measured at OD=550 nm.

DspB, which is a surfacing active against E. coli biofilms, served as anegative control since it has no activity against P. aeruginosa. Twopayloads (Pp15gp44 and SePSMa) showed marked anti-biofilm activity (FIG.7B). Notably, PSMs, which are surfactins with known anti-biofilmactivity in Gram-positive bacteria, have never been previously shown todisperse P. aeruginosa biofilm. These payloads were engineered into WHRLUZ19 to determine if a phage with wide host range can be furtherengineered to display biofilm-dispersing activity. The results show thatWHR LUZ19 encoding Pp15gp44 or SePSMa maintain their biofilm-dispersingactivity (FIG. 7D) and the ability to infect all the clinical isolatessusceptible to the Φ-KMV genus of viruses (FIG. 7E, 7F).

These data provide an example of how the herein described in vitroengineering method can be used in a system to iteratively engineerbacteriophage with multiple independent improved viral properties, suchas the non-limiting properties of biofilm dispersion and host range.

Example VII Engineered Viruses Expressing Antibiotic SensitizingPayloads

Using the herein disclosed in vitro engineering method, LUZ19 wasengineered to express lysins from ssRNA viruses PRR1 and MS2. Lysinsfrom either PRR1 (SEQ ID NO:20) or MS2 (SEQ ID NO:19) ssRNA phage wereengineered into the LUZ19 gp49 locus (SEQ ID NO:25) flanked by the majorcapsid promoter P_(gp32) (SEQ ID NO:21) and terminator T_(gp32) (SEQ IDNO:22) to determine their ability to inhibit emergence of bacteriaresistant to phage (FIG. 8A). These lysins inhibit new cell wallformation by binding and inactivating enzymes important for cell wallsynthesis and putatively sensitize bacteria to other antimicrobials,especially cell-wall targeting antibiotics such as carbenicillin.

The construct was made as described above using the herein disclosed invitro engineering method. Engineered phage were amplified within theirappropriate host cell, isolated, and verified by sequencing. Engineeredphage ability to inhibit the emergence of bacteria resistant to phagetreatment in the presence of carbenicillin at ⅕×MIC was tested in astandard time kill assay (FIG. 8B, 8C). The results show that engineeredLUZ19 expressing lysins from ssRNA phage in combination withcarbenicillin at sub-inhibitory concentrations (⅕×MIC) prevent bacterialre-growth after phage treatment.

These data provide an example of employing the herein disclosed in vitroengineering method to generate a virus with improved viral properties,specifically in this case, prevention of phage-resistance development inbacteria.

Example VIII Engineered Virus Expressing Species-Specific AntimicrobialProtein Payload

Using the herein disclosed in vitro engineering method, LUZ19 wasengineered to express the P. aeruginosa derived antimicrobial proteinPyoS5. The bacteriocin PyoS5 is a species specific antimicrobialproteins produced by one strain of P. aeruginosa to impede the growth ofcompeting P. aeruginosa strains. P. aeruginosa strain PA01 gDNA was usedas template to PCR amplify pyoS5 (SEQ ID NO:6) prior to cloning into theLUZ19 gp49 locus (SEQ ID NO:25) flanked by the major capsid promoterP_(gp32) (SEQ ID NO:21) and terminator T_(gp32) (SEQ ID NO:22) (FIG.9A). PyoS5 binds to the widely dispersed pyochelin receptor FptA beforeundergoing conformational changes to create pores within the P.aeruginosa membrane.

LUZ19+pyoS5 was created as described above using the herein disclosed invitro engineering method. Engineered phage were amplified within thesusceptible host PA01, isolated, and verified by sequencing. Bacterialstrain PA7416 was chosen for analysis because laboratory strain PA01 isknown to be resistant to PyoS5, however, in silico analysis indicatedthe MDR P. aeruginosa strain PA7416 was both susceptible to phage LUZ19and encoded the PyoS5 receptor FptA.

Engineered phage ability to inhibit the emergence of PA7416 bacteriaresistant to phage treatment was tested in a standard time kill assays.The results show that while wild type LUZ19 initially inhibits PA7416growth, bacteria rapidly become resistant and re-growth occurs after8-12 h (FIG. 9B). However, engineered LUZ19+pyoS5 inhibits PA7416bacterial re-growth for greater than 24 h after phage treatment (FIG.9C, 9D).

These data provide an example of employing the herein disclosed in vitroengineering method to generate a virus with improved viral properties,specifically in this case, prevention of phage-resistance development inbacteria.

Example IX System for Iterative Engineering Bacteriophage to Create anAntimicrobial Product

Using the herein disclosed in vitro engineering method, bacteriophagegenomes can be rapidly engineered without extensive genetic manipulationof the host strain. Coupling viral mutation studies and selectiontechniques well known to those in the art, with full genome sequencing,comparative genomics, and the disclosed in vitro engineering methodcreates a new and improved system for developing novel and improvedantimicrobials. The system is based on iteratively improving 1, 2, orgreater than 2 distinct properties in a single viral chassis to create aviral based antimicrobial. The sequential purification and editing ofthe LUZ19 genome to improve distinct viral properties is disclosed(FIGS. 6, 7, and 10), however, this technique could be extended tomultiple other P. aeruginosa bacteriophage or other bacteriophageinfecting any other strain or species of bacteria. Additionally, thistechnique could be used to improve the properties of multiple individualbacteriophage infecting the same bacterial species to create a superiorbacteriophage cocktail preventing or treating bacterial infections,contamination, or to alter a microbiome.

These data demonstrate how in vitro engineering coupled with genomesequencing, comparative genomics, and viral mutation/selection studiescan be performed sequentially to accomplish step-wise improvements orengineered changes to incorporate improved viral properties of interest(FIG. 10).

Example X Methods

Guide RNAs (gRNAs) were synthesized and purified using a commerciallyavailable in vitro transcription kit, such as MEGAshortscript T7 kit(Thermo Fisher). Guide RNAs were designed using methods well known inthe art (FIG. 15).

Dilute in vitro transcribed gRNAs to a working stock of 500 ng/μL.

Assemble reactions without purified RNA-guided nuclease, such as Cas9.Purified Cas9 (SEQ ID NO:31) was obtained from expressing a plasmidcomprising a gene sequence encoding a His-tagged Cas9 (SEQ ID NO:27) andpurifying it through well-known nickel-affinity purification methods.Optionally use gRNA that cuts on the inner-most portion of the genomefirst for iterative digestions.

Full Reaction Mix:

μl 10X Cas9 buffer** 4 50 mM MgCl₂ 8 100 mM Spermidine 4 gRNA 1 2 gRNA 22 Cas9 enzyme (0.45 mg/m1) 8 (total) gDNA X (2 μg total) dH₂O X (to 40uL total volume) *The Full Reaction mix can be used in a single step tocut multiple sites at once (co-digestion), however, this can result inlow efficiency cutting of viral gDNA. Co-digestion reactions areassembled on ice prior to addition of Cas9 and incubation at 37° C. for30 minutes. A modified 2 step (or more) reaction can also be performed,allowing for more complete digestion (outlined below). **10x Cas9 buffercontains- 200 mM HEPES pH 7.4, 1.5M KCl, 5 mM DTT, and 1 mM EDTA pH 8.Assemble Reaction Step 1 and incubate at RT for 5 minutes.Step 1 Reaction Mix:

μl 10X Cas9 buffer 4 50 mM MgCl₂ 8 100 mM Spermidine 4 gRNA 1 2 gDNA 2μg total) dH₂O X (to 36 μl total volume)

Incubate on ice for 10 minutes.

Incubate at 37° C. for 2 minutes.

Add 4 μl Cas9 enzyme (0.45 mg/ml). Incubate at 37° C. for 30 minutes.

Step 2 reaction, addition of second gRNA and additional Cas9 enzyme.

Step 2 Reaction Mix:

μl Step I reaction mixture 40 gRNA 2 2 Cas9 enzyme (0.45 mg/ml) 4 10XCas9 buffer 1 dH2O 3 50 μL total volume

Incubate Step 2 Reaction at 37° C. for 30 minutes. Additional steps canbe added for digesting the genome at more than 2 locations.

Inactivate Cas9 enzyme by incubating at 80° C. for 10 minutes. Optionalpurification using phenol-chloroform extraction (increases efficiency offragment assembly in Gibson Assembly), or other inactivation,deactivation, or purification methods well known in the art.

Run 5 μL of sample on agarose gel to verify proper cutting.

For in vitro assembly using Gibson Assembly, appropriate concentrationof digest and in vitro generated insert DNA were used according to NEBGibson Assembly protocol.

Following in vitro assembly, optionally transform into host cells toamplify engineered genome, genome section, or recover engineered virus.

Example XI Engineering of E. coli Phage M13

Using the herein disclosed in vitro engineering method, a virusinfecting Escherichia coli was engineered to express the fluorescentreporter paprika (SEQ ID NO:5). FIG. 11A shows a schematic of the invitro engineering approach for incorporating the paprika fluorescentprotein gene into the E. coli M13 phage genome. This engineering processwas designed to generate a fluorescent reporter expressing lysogenicphage, which would constitute an improved viral property, as similarviruses have been used as diagnostics. The M13 viral genome (Accessionnumber X02513) was isolated from viral particles. Since the experimentaldesign involves the use of two gRNAs, the functionality of eachindividual gRNA was first confirmed in separate in vitro Cas9 digestionreactions (FIG. 11B). Knowing each gRNA was functional, site-specificdigestion was performed using an RNA-dependent nuclease and both invitro transcribed gRNAs (FIG. 11C). The fluorescent reporter genepaprika (SEQ ID NO:29) was PCR amplified (FIG. 11D) using primers thatadded 5′ and 3′ sequences homologous to the sequences flanking the LacZagene, which was liberated from the M13 genome using RNA-dependentnuclease digestion, for example, Cas9. The Gibson Assembly method wasused to integrate the PCR amplified paprika gene seamlessly into thedigested M13 genome, replacing the LacZa gene (SEQ ID NO:28). Theengineered genomes were transformed directly into host E. coli cells toyield functional viral particles encoding the paprika gene. Engineeredphage were assessed by their ability to form plaques in E. coli (FIG.11E). Viral DNA was isolated from the plaques and PCR amplified toconfirm the presence of the inserted paprika gene (FIG. 11F). Thepresence and function of the recombinant paprika protein was confirmedby fluorescent imaging (FIG. 11G).

These data demonstrate the successful use of the herein described invitro engineering method to engineer a reporter gene into an E. coliphage genome. Demonstrating that the disclosed method is extendable toanother genus of viruses, including those that infect another genus ofbacteria.

Example XII Engineering of E. coli Phage

Using the herein disclosed in vitro engineering method, a second virusinfecting Escherichia coli was edited. FIG. 12A shows a schematic of thein vitro engineering approach to delete the cll gene (SEQ ID NO:30) fromthe isolated λ phage genome (Accession NC_001416.1). This engineeringprocess was designed to generate a constitutively lytic virus, whichwould constitute an improved viral property. The λ viral genome wasisolated from viral particles. Since the experimental design involvesthe use of two gRNAs, the functionality of each individual gRNA wasfirst confirmed in separate in vitro Cas9 digestion reactions (FIG.12B). Knowing each gRNA was functional, site-specific digestion wasperformed using an RNA-dependent nuclease and both in vitro transcribedgRNAs (FIG. 12C). Two synthesized single strand DNA molecules wereannealed in vitro to generate the double stranded DNA repair template(SEQ ID NO:9) comprising 5′ and 3′ sequences homologous to the sequencesflanking the Cas9-targeted cut sites in the isolated λ viral genome. TheGibson Assembly method was used to integrate the PCR amplified repairtemplate seamlessly into the digested λ genome. The engineered genomeswere then packaged in vitro using the Maxplax lambda packagingextraction kit from EpiCentre according to the manufactures method (FIG.12D). Following in vitro packaging, engineered λ genomes were recoveredfrom double agar plaque assays using manufacturer suggested E. coli hostcells. The engineered phage were determined to be functional based ontheir ability to form plaques in E. coli. Viral DNA was isolated fromthe formed plaques and PCR amplified to confirm the absence of the cllgene (FIG. 12E).

These data demonstrate the successful use of the herein described invitro engineering method to remove an unwanted gene from an E. coliphage genome. These data also provide an example of packaging engineeredviral genomes in vitro, which increased the virus recovery efficiencyand provides an alternative to direct transformation into a host cell.Additionally, these data provide an example of utilizing annealed invitro synthesized oligonucleotides as the insert for engineering.Furthermore, these data provide another example of utilizing thisapproach to engineering a phage genome to result in an improved viralproperty, namely a constitutively lytic phenotype. Lastly, these dataindicate that a second genus of virus infecting E. coli can beengineered using the described in vitro engineering method.

Example XIII Error Correction of Human CMV

Using the herein disclosed in vitro engineering method, a portion of ahuman virus was edited. FIG. 13A shows a schematic of the in vitroengineering approach being utilized for error correction. An 18 kbsubsection of the ˜230 kb HCMV viral genome was contained within an E.coli replicating plasmid. This subsection of the HCMV genome (SEQ IDNO:10) contained the start of the viral genome and harbored a mutantRL13 allele (SEQ ID NO:33). Together the HCMV fragment and E. coliplasmid were of roughly 28 kb in size, exceeding the specifications ofmost current error correction techniques. For error correction, the 28kb plasmid was isolated from E. coli and site-specific digestion wasperformed using an RNA-dependent nuclease and two in vitro transcribedgRNAs (FIG. 13B). The Cas9 mediated digestion excised a region of theRL13 gene directly upstream and downstream of the mutation site. Thecorrected region of the RL13 gene (SEQ ID NO:32) was synthesized and PCRamplified with additional 5′ and 3′ flanking sequences homologous to theregions bordering each RNA-specified Cas9 digestion site (FIG. 13C). TheGibson Assembly method was used to integrate the synthesized repairtemplate seamlessly into the digested plasmid. The corrected RL13containing HCMV fragment (SEQ ID NO:11) contained within the plasmid wasthen transformed into E. coli cells and recovered on antibioticcontaining media. E. coli colonies were screened by PCR to confirm thepresence of the corrected RL13 gene, which contained additional sequencecompared to the error-containing RL13 gene, thereby allowing it to bedistinguished from the error-containing RL13 gene (FIG. 13D). The errorcorrected genomic fragment was then amplified in E. coli using standardtechniques, for later use in downstream applications.

These data demonstrate the successful use of the herein described invitro engineering method to engineer genes from a human-specific virusgenome and additionally provides a method for using synthesized DNA asthe repair template in the in vitro assembly reaction. These data alsodemonstrate the use of this in vitro engineering method for errorcorrection of DNA or plasmids that are too large for standard errorcorrection techniques. Standard error correction technique have a sizerestriction around 5 kb and are PCR-based, which inherently can producemore unwanted errors. The herein presented in vitro engineering methoddoes not rely on PCR amplification of the whole or even a large portionof the plasmid or viral genome and therefore is amenable to errorcorrection applications of sequences exceeding 5 kb in size.

Example XIV Rapid Identification of Terminally Redundant Viral Ends

The herein disclosed in vitro digestion method can also be adapted toidentify the exact termini of terminally redundant viral genomes. FIG.14 shows a schematic of the in vitro digestion approach that was used todetermine the ends of LBL3 and 14-1 phage genomes. LBL3 and 14-1(Accession number NC_011703.1) phage genomic DNA was purified from viralparticles (FIG. 14A). Next generation sequencing was performed using theMiSeq or PacBio platform followed by automated merging of the highquality DNA reads into longer assemblies to reconstruct the originalsequence (FIG. 14B). Normally, the automated assembly softwareincorrectly assembles viral or bacteriophage genomes into circularcontigs and places the DTRs of the terminally repetitive genomes in theinternal region of the viral sequence. In silico prediction of thephysical genome ends is performed based on the identification of doublecoverage sequencing regions and BLAST search results that match to aclosely related terminally repeated genome (FIG. 14C). These predictedends were confirmed by Cas9 endonuclease cleavage. After Cas9inactivation, DNA fragments corresponding to the genomic physical endswere purified and sequenced (FIG. 14D). These sequencing results led toan accurate genome assembly based on the true physical end sequences(FIG. 14E).

One of the biggest technical challenges associated with phage genomesequencing is accurate mapping of genomic physical ends due to theirrepetitive nature. These segments can span from 4-14 bp in circularlypermuted genomes (e.g. most Mycobacterium and Propionibacterium acnesphage) to several hundred base pairs in terminally repetitive genomes(e.g. ΦKMV-like, PB1-like and N4-like phage genera of P. aeruginosa) andeven to several thousand base pairs (e.g. E. coli T5 and DTRs). Mappingof repetitive ends (or DTRs—direct terminal repeats) currently isperformed by a combination of in-depth sequence analysis (to identifydouble coverage DNA fragments), primer walking (Sanger sequencing),identification of major DNA nicks, and restriction endonucleaseanalysis. However, each of these approaches are often limited in use orinconclusive do to: (i) poorly defined double sequencing coverageboarders within NGS data; (ii) primer walking reading through DTRconcatamers giving inconclusive results; (iii) low incidence ofrestriction sites near phage termini or obstruction of restriction sitesdue to DNA modifications, such as methylation. The use of targeted Cas9cleavage of phage DNA at specific positions eliminates the need forunreliable or cumbersome analyses or procedures, and greatly simplifiesthe identification of phage genomic physical ends. This approach has thepotential to accurately map the ends of already sequenced phage genomes(as exemplified by the mapping of LBL3 and 14-1 DTRs) as well as rapididentification of DTR of newly identified viruses.

Using targeted Cas9 digestion within the herein disclosed in vitroengineering method to map the physical ends of terminally repetitivephage genomes represents a distinct advantage over the currentapproaches because it does not rely on subtle changes in sequencingcoverage and can be performed independent of concatemer formation. Inaddition, Cas9 activity is less sensitive to DNA modifications than manyrestriction enzymes.

These data show the successful employment of RNA guided in vitro Cas9cleavage to enable the identification of true phage genome sequencearrangement. This information can then be used to design downstream invitro engineering approaches to engineer these phage, a feat that waspreviously impossible due to the lack of a true genome boundaries.

Example XV Engineering Method with In Vivo Assembly

The present disclosure provides for an in vitro method ofsite-specifically digesting a purified viral nucleic acid using anRNA-guided nuclease; and assembling an engineered nucleic acid by theinsertion of a DNA or RNA fragment into the digested viral nucleic acid.While the recombinant nucleic acid can be assembled completely in vitroutilizing purified enzymes as disclosed herein, this process can also beaccomplished utilizing natural or engineered recombination pathwayswithin a susceptible host strain. Transformation of purified and invitro digested viral genomes along with an insert repair fragmentharboring terminal homology regions is sufficient for some host cells toassemble a recombinant viral genome in vivo. Insert repair fragments canbe synthesized or amplified by standard techniques known in the art orcan reside within plasmids stably replicating within the chosen hostcell. This method is likely to have lower efficiency than in vitroassembly due to host cells having both homologous and non-homologous DNArepair pathways, the challenge of co-delivering sufficient quantities ofinsert and digested genome into a host cell, and the lower efficiency ofmost host homologous recombination pathways. As digested genomes alonewill not form functional viral particles and subsequent plaques withouthost-mediated recombination, the plaques obtained followingtransformation and plating can be screened by PCR for the given insertto confirm correct assembly of the desired engineered viral nucleicacid.

Example XVI Engineered Viruses Disclosed Herein

Table 1 summarizes the engineered viruses generated through the hereindisclosed in vitro engineering method. Table 2 summarizes the engineeredviruses disclosed herein along with the corresponding Example andFigure. Table 3 lists the wild type viruses disclosed herein and theAccession numbers for their full genomic sequence. Table 4 lists some ofthe wild type nucleic acid sequences disclosed herein and thecorresponding amino acid sequences.

TABLE 1 Engineered Viruses disclosed herein Nucleotide and aminoStarting Mutation Accession No., nt position (SEQ acid change orsequence Engineered Virus Virus type Region target ID NO.) added LUZ19 +ΦKF77 LUZ19 Replace gp7 ACCESSION NC_010326.1, 4288-4491 ΦKF77 gp7 frag.gp7 fragment (SEQ ID NO: 23) (SEQ ID NO: 23) (SEQ ID NO: 8) LUZ19 +LKD16 LUZ19 Replace gp18 ACCESSION NC_010326.1, LKD16 gp18 gp18 (SEQ IDNO: 50) 11368-11688 (SEQ ID NO: 24) (SEQ ID NO: 7) WHR LUZ19 LUZ19 PMgp13 ACCESSION NC_010326.1, 7325 G to A, C17Y (SEQ ID NO: 1) (pos. 50 ofSEQ ID NO: 1) PM gp18 ACCESSION NC_010326.1, G to T, D36Y (SEQ ID NO:50) 11469 (pos. 106 of SEQ ID NO: 50) PM gp38 ACCESSION NC_010326.1, Ato G, D82G (SEQ ID NO: 2) 36462 (pos. 245 of SEQ ID NO: 2) PM gp38ACCESSION NC_010326.1, AT to TC, I83S (SEQ ID NO: 2) 36464; 36465 (pos.247, 248 of SEQ ID NO: 2) PM gp40 ACCESSION NC_010326.1, A to G, N253D(SEQ ID NO: 3) 38180 (pos. 757 of SEQ ID NO: 3) LUZ19 + gp34 LUZ19Delete gp34 ACCESSION NC_010326.1, CTG to —, L55 L55Δ (LUZ19*) (SEQ IDNO: 4) 26664-26666 (pos. 163-165 of SEQ ID NO: 4) LUZ19 + LKD16 LUZ19 +LKD16 Delete gp34 ACCESSION NC_010326.1, CTG to —, L55 gp18 + gp34 L55Δgp18 (SEQ ID NO: 4) 26664-26666 (pos. 163-165 of SEQ ID NO: 4) LUZ19 +LKA1gp49 LUZ19 Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO: 21)(SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) LKA1 gp49 (SEQ ID NO: 12)T32 (SEQ ID NO: 22) LUZ19 + NTUgp34 LUZ19 Insert gp49 ACCESSIONNC_010326.1, P32 (SEQ ID NO: 21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO:25) NTUgp34 (SEQ ID NO: 13) T32 (SEQ ID NO: 22) LUZ19 + Pp15gp44 LUZ19Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO: 21) (SEQ ID NO: 51)42719-42943 (SEQ ID NO: 25) Pp15gp44 (SEQ ID NO: 14) T32 (SEQ ID NO: 22)LUZ19 + SaPSMa3 LUZ19 Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO:21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) SaPSMa3 (SEQ ID NO: 16)T32 (SEQ ID NO: 22) LUZ19 + SaPSMb2 LUZ19 Insert gp49 ACCESSIONNC_010326.1, P32 (SEQ ID NO: 21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO:25) SaPSMb2 (SEQ ID NO: 17) T32 (SEQ ID NO: 22) LUZ19 + SePSMa LUZ19Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO: 21) (SEQ ID NO: 51)42719-42943 (SEQ ID NO: 25) SePSMa (SEQ ID NO: 18) T32 (SEQ ID NO: 22)LUZ19 + dspB LUZ19 Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO:21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) dspB (SEQ ID NO: 15) T32(SEQ ID NO: 22) LUZ19 + Pp15gp44 WHR Insert gp49 ACCESSION NC_010326.1,Pp15gp44 LUZ19 (SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) (SEQ ID NO:14) LUZ19 + SePSMa WHR Insert gp49 ACCESSION NC_010326.1, SePSMa LUZ19(SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) (SEQ ID NO: 18) LUZ19 + PPR1L LUZ19 Insert gp49 ACCESSION NC_010326.1, P32 (SEQ ID NO: 21) (SEQ IDNO: 51) 42719-42943 (SEQ ID NO: 25) PRR1 L (SEQ ID NO: 20) T32 (SEQ IDNO: 22) LUZ19 + MSR L LUZ19 Insert gp49 ACCESSION NC_010326.1, P32 (SEQID NO: 21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO: 25) MS2 L (SEQ ID NO:19) T32 (SEQ ID NO: 22) LUZ19 + pyoS5 LUZ19 Insert gp49 ACCESSIONNC_010326.1, P32 (SEQ ID NO: 21) (SEQ ID NO: 51) 42719-42943 (SEQ ID NO:25) pyoS5 (SEQ ID NO: 6) T32 (SEQ ID NO: 22) M13MP18 + paprika M13MP18Replace lacZ ACCESSION X02513, 6216-6722 paprika (SEQ ID NO: 28) (SEQ IDNO: 28) (SEQ ID NO: 29) Lambda c// Lambda Delete c// ACCESSIONNC_001416.1, c// deleted deletion (SEQ ID NO: 30) 38390-28623 (SEQ IDNO: 30) (SEQ ID NO: 9) HCMV + edited Human Replace RL13 Unedited fulllength fragment Edited RL13 (SEQ ID RL13 CMV (SEQ ID NO: 33) (SEQ ID NO:10) and unedited NO: 32) and edited full Fragment RL13 (SEQ ID NO: 33)length fragment (SEQ ID NO: 11) PM—point mutation, Replace—replacement,Delete—deletion, Insert—insertion

TABLE 2 Engineered viruses disclosed herein Engineered Phage ExampleFIG. Property LUZ19 + ΦKF77 gp7 I 2 Engineering POC fragment LUZ19 +LKD16 II 3 Host Range Expansion gp18 WHR LUZ19 III 4 Host RangeExpansion LUZ19 + gp34 L55Δ IV 5 Improved Lytic Activity (LUZ19*)LUZ19 + LKD16 V 6 Iterative Engineering gp18 + gp34 L55Δ LUZ19 +LKA1gp49 VI 7 Biofilm Dispersion LUZ19 + NTUgp34 VI 7 Biofilm DispersionLUZ19 + Pp15gp44 VI 7 Biofilm Dispersion LUZ19 + SaPSMa3 VI 7 BiofilmDispersion LUZ19 + SaPSMb2 VI 7 Biofilm Dispersion LUZ19 + SePSMa VI 7Biofilm Dispersion LUZ19 + dspB VI 7 Biofilm Dispersion WHR VI 7Iterative Engineering LUZ19 + Pp15gp44 WHR VI 7 Iterative EngineeringLUZ19 + SePSMa LUZ19 + PPR1 L VII 8 Antibiotic Sensitization/PhageResistance Prevention LUZ19 + MS2 L VII 8 Antibiotic Sensitization/PhageResistance Prevention LUZ19 + pyoS5 VIII 9 Phage Resistance PreventionM13MP18 + paprika XI 11 Engineering POC λ c// deletion XII 12Engineering POC HCMV + edited XIII 13 Error RL13 Correction/EngineeringPOC POC—proof of concept

TABLE 3 Wild type viruses disclosed herein Wild type virus name GenomicSequence P. aeruginosa phage LUZ19 ACCESSION NC_010326.1 E. coli phage λc// 857 SAM7 ACCESSION NC_001416.1 E. coli phage M13 ACCESSION X02513 P.aeruginosa phage 14-1 ACCESSION NC_011703.1

TABLE 4 Wild type sequences disclosed herein Name Nucleic acid sequenceAmino acid sequence LUZ19 gp13 SEQ ID NO: 1 SEQ ID NO: 34 LUZ19 gp38 SEQID NO: 2 SEQ ID NO: 35 LUZ19 gp40 SEQ ID NO: 3 SEQ ID NO: 36 LUZ19 gp34SEQ ID NO: 4 SEQ ID NO: 5 LUZ19 gp49 SEQ ID NO: 51 SEQ ID NO: 49 LUZ19gp18 SEQ ID NO: 50 SEQ ID NO: 48 LKD16 gp18 SEQ ID NO: 26 SEQ ID NO: 38LKA1 gp49 SEQ ID NO: 12 SEQ ID NO: 39 PyoS5 SEQ ID NO: 6 SEQ ID NO: 37NTU gp34 SEQ ID NO: 13 SEQ ID NO: 40 Pp15 gp44 SEQ ID NO: 14 SEQ ID NO:41 DspB SEQ ID NO: 15 SEQ ID NO: 42 SaPSMa3 SEQ ID NO: 16 SEQ ID NO: 43SaPAMb2 SEQ ID NO: 17 SEQ ID NO: 44 SePSMa SEQ ID NO: 18 SEQ ID NO: 45MS2 L SEQ ID NO: 19 SEQ ID NO: 46 PRR1 L SEQ ID NO: 20 SEQ ID NO: 47

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this disclosure, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the disclosure to adapt it to various usage andconditions and to utilize the present disclosure to its fullest extent.The preceding specific embodiments are to be construed as merelyillustrative, and not limiting of the scope of the disclosure in any waywhatsoever. The entire disclosure of all applications, patents, andpublications (including reference manuals) cited above and in thefigures, are hereby incorporated in their entirety by reference.

SEQUENCE LIST SEQ ID NO: 1 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp13GTGCTGGCCCTCGGTGCCTTCGACCTGTCCGGCCTGATGGTAGGTTCCTGCCTCGTAGTAGGTGGTGAGCTGAAGGCCCTGTGCGTTGATGACCGGCACAGCAGGCAGGGTATCGGCGCTGAGCTGGTACGGGCCGCTGAGCTGGCTGGTGCCGAGTATCTGACCTGCTTCGAGTTCCTGGAGCCGTTCTACGCCGACTTGGGCTGGAGCACCACCCACCGCGAGGCGAACTGGACAGCAGGAGAGCCGGACGTGCTGCACATGAGGGCACCCGGTCATGACGTATGASEQ ID NO: 2 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp38GTGGCTCGGTTCAAGAATCCCGAGACCATCCACGTTGCAGATGGGGTCGAGGCTGTCTTCAGTCTCGACTTCCCGTTCCTGCGGCGTGAGGACGTATTCGTCCAGGTCGATAAGATACTCGTCACCGACTATACGTGGGTAGACGACACCAACATCCAATTGGCCGTGGTGCCGAAGAAGGACCAAGAGGTCCGCATCTTCCGCGACACGCCCGCCCAGGTCCCGGACACACAGTTCAGCCAGGACATCCCGTTCCTGCCTCGATACATCGACGCGAACAACAAGCAGCTCCTGTACGCTGTGCAGGAAGGCATCAACACCGCGAACCTCGCTCTCGATGGCGTACTCGACGCGATCCGTATCGCCGAGGAGGCTCGTCGCCTGGCGCAGGAAGCACTCGACGCCGCCAATGAGGCGCTTCGCCGTGCCCTGGGCTTCGCTGAGATTCGCACCGTGACCGAGGACTCGGACATCGATCCGAGCTGGCGCGGTTACTGGAACCGTTGCATCACCGCCGATAAACCTCTGACCCTGACCATGCAGATGGAAGACCCGGATGCACCGTGGGTCGAGTTCAGCGAGGTTCACTTCGAGCAGGCCGGTGTGCGTGACCTAAACATCGTAGCCGGTCCTGGCGTTACCATCAACCGTTTGCAGAACACCACCATGCAGCTCTACGGCGAGAATGGCGTGTGTACTCTCAAGCGGCTGGGCGCTAACCACTGGATCGTGTTCGGGGCCATGGAGGACGAATAA SEQ ID NO: 3 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp40ATGTTTAAGACCGAAGTAAAGGGACGTTACACCCTGATTCGCCGCAAGGCGGACGGCACTCCGGTGGAGACTCTGGAGTTCGACAACATCATTACGAATGCGGGCCTGGATTGGATCGCCGCTATGGATACCGACCTCATGGGCGAACCCGTAGCGGTCAGCACTTCTACAGCCGATCCCAACCCGAGCGCACCCGCCATCCCGGAGGTTGTGCAACGCACGTCCGCATCTGCCCCTGGTGGAGGTACTACGTCGGGCCTGGATGGCGAGTGGCTGTTCTGGCGGAGGCGTTGGAGATTCCCGCAGGGCACCCTAGCTGGTCAAGTCCTGGCCACCGTGGGCCTCATCTGCAACTCGGATCGTCGCTTCGAGAGTAACACGGGTGAGCTGATCCCGAAGGATACCCCGCTGTCGTACACTCGCATCAAGGACGCCGCCGGGCAGCCTACTACTCTGGTGGTGGCCGCTGACGAGATTCTGGATGTCCAGTACGAGTTCCGCAGCCGGCCCGTAGGAACGGCTGAGGCCAAGTTCGTGATCTCCGGCGTGGAACGCACCTTCCGGCTGATCCCAAAGCCTTTTGCGAACCGTGCTAATCTCTCCGGGGAACGCTACATCTTCTACAACACCAACCCCTACATCAACGGCAAGGACGCCTCCGGCGGCAATGTCCGAGACGGTCAGTGGCAGAAGAAATATCCCAAGTACGTGCGCGGCTCCTACAAGGCGCAGATCACGCTGCTGGCCCAGGTCCAGAACGGCAATATGGCTGGCGGCATCACCGGCACCGAGGAACTCCAGATTTACAATGGACGTAACTATGTGCTCGATATCAACCCGCCTGTTGTGAAGAACAATACCCAGGAGTTCACCGTGACCCTGGAGTTTACGGTGGCGAGGGCATAA SEQ ID NO: 4 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp34ATGAGCTACAAGCAATCCGCGTATCCCAATCTGCTGATGGGTGTGAGCCAGCAGGTGCCCTTCGAGCGCCTGCCGGGCCAGCTCAGCGAGCAGATCAACATGGTATCCGATCCCGTGTCAGGACTTCGGCGGCGCAGCGGTATCGAGCTGATGGCCCACCTGCTGCATACCGACCAGCCCTGGCCGAGGCCGTTCCTCTACCACACGAACCTCGGTGGCCGCAGCATTGCGATGCTGGTGGCGCAGCACCGTGGCGAGCTGTACCTGTTCGACGAGCGGGACGGTCGCCTGCTGATGGGTCAGCCCCTGGTGCATGACTACCTCAAGGCCAACGATTACAGGCAGCTACGGGCCGCCACGGTGGCCGATGACCTGTTCATCGCCAACCTGAGTGTAAAGCCCGAGGCCGACCGCACCGACATCAAGGGCGTAGACCCCAACAAGGCCGGCTGGCTGTACATCAAGGCAGGCCAGTATTCGAAGGCATTCTCCATGACCATCAAGGTCAAGGACAACGCCACCGGCACCACCTACAGCCACACGGCCACCTACGTGACGCCGGACAACGCCAGCACGAACCCCAACCTCGCTGAGGCGCCATTCCAAACGAGCGTAGGCTACATCGCGTGGCAGCTCTACGGCAAGTTCTTCGGTGCGCCGGAGTACACTCTGCCCAACTCGACGAAGAAGTACCCGAAGGTAGACCCGGACGCCAACGCGGCAACCATAGCCGGTTACCTCAACCAACGGGGCGTGCAGGACGGGTACATCGCGTTCCGTGGCGACGCCGATATCCACGTTGAAGTGTCCACGGACATGGGCAACAACTACGGCATAGCCTCCGGCGGTATGAGCCTCAACGCCACGGCAGACCTGCCGGCCTTACTGCCGGGCGCGGGTGCTCCTGGCGTGGGTGTGCAGTTCATGGACGGCGCTGTCATGGCCACCGGCTCCACCAAGGCCCCGGTATACTTCGAGTGGGATTCCGCTAACCGCCGCTGGGCAGAGCGGGCCGCCTACGGCACCGATTGGGTCCTGAAGAAGATGCCACTGGCCCTGCGCTGGGATGAGGCTACCGACACCTACAGCTTGAACGAGCTGGAGTATGATCGACGTGGCTCCGGCGACGAGGATACGAACCCCACGTTCAACTTCGTCACCCGAGGCATCACCGGCATGACGACCTTCCAGGGTCGCCTCGTCCTCCTGTCGCAGGAGTACGTCTGCATGTCGGCCAGTAACAATCCACACCGCTGGTTCAAGAAGTCGGCAGCCGCGCTGAACGACGATGATCCTATCGAGATCGCAGCCCAGGGGAGCCTGACTGAACCGTACGAGCACGCGGTCACCTTCAACAAGGACTTGATCGTCTTCGCCAAGAAGTATCAGGCCGTGGTCCCCGGTGGCGGCATTGTAACTCCCCGGACGGCGGTTATCAGCATCACCACGCAGTACGACCTCGATACCAGGGCGGCACCTGCCGTGACTGGCCGCAGTGTGTACTTCGCTGCGGAGCGTGCCCTGGGTTTCATGGGCCTGCATGAGATGGCCCCGTCTCCGTCCACGGACAGCCACTACGTCGCCGAAGACGTTACCAGCCACATCCCGAGCTACATGCCGGGGCCTGCTGAGTACATCCAGGCGGCGGCCTCCAGCGGCTACCTGGTGTTCGGCACCAGCACGGCGGACGAGATGATCTGCCACCAGTACCTCTGGCAGGGCAACGAGAAAGTGCAGAACGCGTTTCATCGCTGGACGTTGCGGCATCAGATCATCGGCGCCTACTTCACTGGTGACAACCTGATGGTTCTGATTCAGAAGGGCCAGGAGATCGCCCTGGGACGGATGCACCTGAACAGCCTGCCAGCCCGTGAGGGTCTGCAATACCCTAAATACGACTACTGGCGGCGTATCGAGGCGACCGTCGATGGTGAGCTGGAACTGACCAAGCAGCATTGGGACCTGATCAAGGATGCCTCTGCCGTGTACCAGCTACAGCCTGTGGCCGGCGCCTACATGGAGCGTACCCATCTCGGCGTGAAGCGCGAGACGAATACGAAGGTGTTCCTCGACGTGCCCGAGGCCGTGGTCGGGGCGGTGTATGTGGTCGGCTGCGAGTTCTGGTCGAAGGTGGAGTTCACTCCGCCGGTTCTCCGGGACCACAATGGCCTGCCCATGACCTCGACCCGTGCAGTGCTTCATCGGTACAACGTAAACTTCGGCTGGACCGGCGAGTTCCTGTGGCGCATCAGCGACACGGCTCGACCCAACCAGCCGTGGTACGACACGACGCCCCTCCGGTTGTTCAGCCGGCAACTCAATGCCGGGGAGCCTCTGGTGGATAGCGCTGTGGTGCCGCTGCCGGCACGGGTCGATATGGCCACGTCCAAGTTCGAGCTGAGCTGTCACAGTCCGTACGACATGAACGTTCGGGCTGTCGAGTACAACTTCAAGTCCAACCAAACCTACAGGAGGGTGTGA SEQ ID NO: 5 Protein Genus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 Gp34 proteinMSYKQSAYPNLLMGVSQQVPFERLPGQLSEQINMVSDPVSGLRRRSGIELMAHLLHTDQPWPRPFLYHTNLGGRSIAMLVAQHRGELYLFDERDGRLLMGQPLVHDYLKANDYRQLRAATVADDLFIANLSVKPEADRTDIKGVDPNKAGWLYIKAGQYSKAFSMTIKVKDNATGTTYSHTATYVTPDNASTNPNLAEAPFQTSVGYIAWQLYGKFFGAPEYTLPNSTKKYPKVDPDANAATIAGYLNQRGVQDGYIAFRGDADIHVEVSTDMGNNYGIASGGMSLNATADLPALLPGAGAPGVGVQFMDGAVMATGSTKAPVYFEWDSANRRWAERAAYGTDWVLKKMPLALRWDEATDTYSLNELEYDRRGSGDEDTNPTFNFVTRGITGMTTFQGRLVLLSQEYVCMSASNNPHRWFKKSAAALNDDDPIEIAAQGSLTEPYEHAVTFNKDLIVFAKKYQAVVPGGGIVTPRTAVISITTQYDLDTRAAPAVTGRSVYFAAERALGFMGLHEMAPSPSTDSHYVAEDVTSHIPSYMPGPAEYIQAAASSGYLVFGTSTADEMICHQYLWQGNEKVQNAFHRWTLRHQIIGAYFTGDNLMVLIQKGQEIALGRMHLNSLPAREGLQYPKYDYWRRIEATVDGELELTKQHWDLIKDASAVYQLQPVAGAYMERTHLGVKRETNTKVFLDVPEAVVGAVYVVGCEFWSKVEFTPPVLRDHNGLPMTSTRAVLHRYNVNFGWTGEFLWRISDTARPNQPWYDTTPLRLFSRQLNAGEPLVDSAVVPLPARVDMATSKFELSCHSPYDMNVRAVEYNFKSNQTYRRV SEQ ID NO: 6 DNAGenus/species-Pseudomonas aeruginosa Descriptive title-PyoS5 sequenceATGTCCAATGACAACGAAGTACCTGGTTCCATGGTTATTGTCGCACAAGGTCCAGACGATCAATACGCATACGAGGTTCCCCCTATCGATAGCGCGGCCGTTGCCGGGAATATGTTTGGCGACTTAATTCAAAGAGAAATATATCTACAGAAAAACATTTATTATCCAGTCCGATCTATTTTTGAACAAGGAACAAAAGAAAAGAAGGAGATCAACAAGAAAGTATCTGATCAAGTCGATGGCTTGCTAAAGCAGATCACTCAAGGAAAAAGGGAGGCCACAAGGCAAGAGCGAGTCGATGTCATGTCGGCAGTCCTGCACAAGATGGAATCTGATCTTGAAGGATACAAAAAGACCTTTACCAAAGGCCCATTCATTGACTACGAAAAGCAGTCAAGCCTCTCCATCTATGAGGCCTGGGTCAAGATCTGGGAGAAGAACTCTTGGGAAGAAAGAAAGAAGTACCCTTTTCAGCAGCTTGTTAGAGATGAACTGGAGCGGGCGGTTGCCTACTACAAACAAGATTCACTCTCTGAAGCGGTAAAAGTGCTAAGACAGGAGCTCAACAAGCAAAAAGCGCTAAAGGAAAAAGAGGACCTCTCTCAACTGGAGCGGGACTACAGAACCCGAAAGGCGAATCTCGAGATGAAAGTACAATCCGAGCTTGATCAAGCGGGAAGTGCTTTGCCTCCATTGGTCAGTCCAACGCCAGAGCAATGGCTTGAACGTGCCACAAGACTGGTTACGCAAGCAATTGCTGATAAAAAGCAGCTGCAGACCACAAACAATACTCTTATCAAGAATTCCCCAACCCCTCTAGAAAAGCAGAAAGCCATCTACAATGGTGAGCTACTTGTGGATGAGATAGCCAGTCTACAGGCCCGCTTAGTTAAGCTGAACGCCGAAACGACACGACGCAGGACAGAAGCAGAACGCAAGGCGGCCGAGGAACAAGCGTTGCAAGATGCTATTAAATTTACTGCCGACTTTTATAAGGAAGTAACTGAGAAATTTGGCGCACGAACATCGGAGATGGCGCGCCAACTGGCCGAAGGCGCCAGGGGGAAAAATATCAGGAGTTCGGCGGAAGCAATCAAGTCGTTTGAAAAGCACAAGGATGCGTTAAATAAAAAACTTAGCCTTAAAGATAGGCAAGCCATTGCCAAAGCCTTTGATTCTCTAGACAAGCAGATGATGGCGAAGAGCCTTGAGAAATTTAGCAAAGGCTTTGGAGTTGTAGGCAAAGCTATTGACGCCGCCAGCCTGTACCAAGAGTTCAAGATATCTACGGAAACCGGGGACTGGAAACCATTCTTTGTAAAAATTGAAACACTAGCTGCTGGTGCGGCCGCCAGTTGGCTTGTGGGTATTGCATTTGCCACGGCAACAGCCACTCCTATAGGCATTCTGGGGTTCGCACTGGTAATGGCAGTTACCGGGGCGATGATTGACGAAGACCTTCTAGAAAAAGCAAACAATCTTGTAATATCCATTTAASEQ ID NO: 7 DNA Genus/species-Phikmvlikevirus LKD16Descriptive title-LKD16 gp18 sequence addedGAGTACCAACTGAACACGAGCGCACCCTGCGCTGCCTGCTCCAAGACATCCACGGGCCGCTGAATCTGCTGTTCCCAGGTATCCGGGTGAAGGTGGAGGAGGCGTGCCTCGGATACTTGGGCTACAGGGAGCGGGGCTATTGGGAGCTGCGCCTCCAGGTGGACTACGACCACCCGAAGCTTGGGCACCTCCGCTACAGTCAGGCCGTGCCGGAGTACGTGCTGATCAACGACCGCGACAGCATCATCAAGTACCTGATGGAAGCAGTCCCTCGGCAGGTACTAGAGGGCATGCTCAATAAGGCCCAGGAATTCGTAACCAAGAACTGGTATTCCCTATGACGAC SEQ ID NO: 8 DNAGenus/species-Phikmvlikevirus phi-KF77Descriptive title-ΦKF77 gp7 sequence addedTACAAGGTGGTGACGCCTAGCTCGGCAGAGGGCGCCGTTGTGCTGGCGACCAAGCAGACGCCTGCCCTCGCTCAGGCAGTCATCGTACTGCACAGCATGAACCCCGCGCAGTACGCGGTGGGCACGGCCATACTAAACACAGACTGGCGGTGCCGCCGCCTGGGTGCCGGCGAGTACATCAAGCTCGTTCAAGGGGAGGCCGAC SEQ ID NO: 9 DNAGenus/species-lambdalike lambda Descriptive title-E. coli phage λ cIIATGGTTCGTGCAAACAAACGCAACGAGGCTCGTTCTGAACAAATCCAGATGGAGTTCTGASEQ ID NO: 10 DNA Genus/species-Cytomegalovirus HCMVDescriptive title-HCMV fragment pre-editingACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGATTACCCTGTTATCCCTACCATTCCGGGCCGTGTGCTGGGTCCCCGAGGGGCGGGGGGGTGTTTTTAGCGGGGGGGTGAAATTTGGAGTCTTGGAGCCGCGTGTGCTGTGGAGGACGGTGACGGTGGTAAGAGTGTGCTGCGGTGCGGTTGGGACGGCGGCGGCGAATAAAAGCGGCGTGCGGCGCGCACGGCGAAAAGCAGACGCGCGTCTGTGTTGTGTGTCTTTGACCGCGGCGGAACACACGCGGAAAAGCGAGTCCCAGGGGACACACGACGAGCGAGTCCCAGGGGGGGACGACGACGGCCAGGGACGCGGAAACGACGCGGAAAAGAGGAAGTCCCCAGGGGGACGGGCGGAAAAGAGGAAGCGCCTAGGGGACCGCGGGGGCAGGAACAGACGAAGTACGCCGCAACCCGCGTCGAGGACACACGCAGAAGCGGCCGCCCAGGGGAGGGGGGGGGGGGGACTCGCGGGCCCCGGGGCACACTTGTTGTTCCCTCCGGCCGCCGACACGCACCCCGAAGCCGCGCACACCGCCGACACACCCCTGACACACCCGCGACACACCCGCCACACGCCCGACACACGCCCGCGACACACCCGACCGACACACCCTGACACACCCCGCCAACACACCCAGCCGCACCCGCCCCGCCAACACACCCCCGACACACCCGACACACGCCCGCGACACACCCGGCACACACCCACCCACCCAGCCGCGCCCCCGACACACCCCGAACGGCGCCGGTGCGGGACAGGGCTCACGGAGGTTTGCGGGCCGTGAGCACGCCTCCCTTTGTACACACTACCGGTGCGTGGCGTCCCACGCTATTTGTTCGCGAGACCGGACTAAGGGAGGTTTGCGGTGCGTCAGCGCGGGGCGGCGTTTGCGGCGTGTTTCGACCAGCGCTTTGTGCGCGCTGCCTGTGCGTGTCGTCCCATGGTCTTTGTCAGCGGCACGGCGCTGGGGACGGGGTTTCACCGCGCTGAGGGATCTTTCTGCGGGTGTGAGGGACGGAGCTTTTTTCGCACGCTGGGCACCGGGCTGGGGGACGGGGGGTGTGCGGGACGGCGGTGGGGCCGGGGCGTTGCGGGTACGGGGATTACGCTGGGAACGGGGACTCGCGGACCCGGGCTGAGGGACGGGGGTGGCGGGGGTGTTTGCGGCGAGGACGGGGGCCTTTTGCGGCGGGGACGGGGACTCACCCTCGCCTATTTAACCTCCACCCACTTCAACACACACATGCCGCACAATCATGCCAGCCACAGACACAAACAGCACCCACACCACGCCGCTTCACCCAGAGTACCAACACACGTTACCCTTACACCACAGCAACACACAACCGCCTATCCAAACCTCGGACAAACACGCCAACGAAGAACACCGCACGCAGATGGAGCTCGACGCCGCGGATTACGCTGCTTGCGCGCAGGCCCGCCAACACCTCTACGCTCAAACACAACCCCAACTACACGCATACCCCAACGCCAACCCTCAGGAAAGCGCTCATTTTTCCACAGAAAATCAACATCAACTCACGCATCTACTTCACAACATTGGCGAAGGCGCAGCGCTCGGCTACCCCGTCCCCCGCGCGGAAATCCGCCGCGGCGGTGGCGACTGGGCCGACAGCGCGAGCGACTTCGACGCCGACTGCTGGTGCATGTGGGGACGCTTCGGAACCATGGGCCGCCAACCTATCGTGACCTTACTGTTGGCGCGCCAACGCGACGGCCTCGCTGACTGGAACGTCGTACGCTGCCGCGGCACAGGCTTTCGCGCACACGATTCCGAGGACGGCGTCTCTGTCTGGCGTCAGCACTTGGTTTTTTTACTCGGAGGCCACGGCCGCCGTGTACAGTTAGAACGTCCATCCGCGGGAGAAGCCCAAGCTCGAGGCCTATTGCCACGCATCCGGATCACCCCCATCTCCACATCTCCACGCCCAAAACCACCCCAGCCCACCATATCCACCGCATCGCACCCACATGCTACGACTCGCCCACATCACACGCTCTTTCCTATCCCTTCTACACCCTCAGCCACGGTTCACAATCCCCGAAACTACGCCGTCCAACTTCACGCCGAAACGACCCGCACATGGCGCTGGGCACGACGCGGTGAACGTGGCGCGTGGATGCCGGCCGAGACATTTACATGTCCCAAGGATAAACGTCCCTGGTAGACGGGGTAGGGGGATCTACCAGCCCAGGGATCGCGTATTTCGCCGCCACGCTGCTTCACCGATATCCAATAAACCCATCCCCTCGCCACGACGTCTCCGCGTATCTTTGTAGCCTCAGGAATCCGTCCCCACGTCCATCCATCCCGAGCACTCCACACGCTATAACAGACCACGGACACGGCAAATGCATGCAAACTTCTCATTTATTGTGTCTACTACTCTGTGTTGCTACAGGGAGTGAAGGGGGTGAAGGCAAAGAAAAAAAAAAGGAACAAAATAATAGATTAGCAGAAGGAATAATCCGTGCGACCGAGCTTGTGCTTCTTTTCTTATAAGGAGGCAAATATACTAGGGAAAACTTAAGAATAGGAAGAAACCGAGGTTTGGGAGAAAAGCTGAGATAAAATAGCGCATTTTCCATACAGAGGTTGTTGTTTTTGTGGATCCTAAGAGGTTTCAAGTGCGAATCTCAAAGTTCTCACGAGAATATTGTCTTCAAGAATCGACAACTGTGGTCCAAGATTTTTTTTTGGTCTTTTTAGGTTCTGCGAGGGACATCACGATGGATCGTTGCGATGAAGTCACGCGTACGCCTCTGGTGTGGCGCGGTGTCGTGACAGGAGAGTGTGTTTTCAGTGCAGAGCTGTCTTGATTCCTATATCCGAGTATCTGTTTTCTCGTAAGGACGGTAATCTTCTTTGGTGTAAGTACATCTAAAAGCTGCAAACTATATTTTAAGGGCTGTCTCTAGGTGTACTTTGATGCTGGAGTTTTTCGCTGTGTTGATGTGAATAAATCTACTACTACTATTATATGCAGAAAGAGTGATTATGCCGAGACAAGATTGCATTGGCTGAACTGTTTCAAAAACGCCTACACTCTACTTATCCGTAAACCTAAGGTAATACTATGTGTAAGTTGTTTTTTTTTCTTTTTGTAGTAAAATGGTGATACGTGCAATTAAAACTGTATTCCATGTTTCCATCCTTTCATTTCAACTTTAAAGGCGGCTTTGAGAGCGAAGAAGTGCGAGGATAAAAATGGATGACTCCTTCGTGTCCAGGGAGTCGACTACTGCAACGCTGATTGATTAAAAGATGGTCTCCGATGATGATGTTGTTATTGATCGAATCATGGTGCAGAACGGCGACGGAGAGGAGCGTGTCCGCCGCCGGGAAGGTGGTCTCTTTCTCTTTTCTTTTTTCAAGAAATCTTCCATGTGTTTATCGTAGTGATCGAAATCGACTGATCTCGGGTTCTTTTTGTTGGTTTCTTTTCGGTTAATCATGTATTGTTTTCTTTTTTTACAGAAAGATACTTTTTTCATGAGCAATTCCTCGCCCGGCGCCGGCATGCCGAGGTGGGGCCACTGCGATCAGCGGCATGCCGACGCCGACCCGGGGATCTTGGATTCACCGTTTTCTCTCTTCTCTCTCTACATACAGACCGGGTGGCAGGAGCGGTAAGGAATCATCGTCGTCTTTCATTCTTCGATGATTATGGTAATACTAAATCTTATCTAGGAGCATATACATCTAAGATTGGAGTACTAGTAGTCGTTTGTGGTTTCTATTTTTTTTATATTTATCTATGACAGTTTTTCTGTTTTTCGTTTTGATAATAATATAATAAAAACTCATGGACGTGAAATCTGGCTTGGTTGTGGTGATTTCATTCTCATTATTGTTGTTTTCTTTCCGTCTTGCGGATGAAGATGTTGCGATGCGGTTGTTGTTGGTGTTGCTATACACCGAGAGAGATGATCTTTTTGTTCTTCTGGTTCATTTCCTATGATTGTTTGGCTGCTGACCGACGCGTCAGGATGTGCAGGGCATGCGGGGAATCAGGACCGGACACGGGATAATTTCATCTACCTATACGGAGATCGCGGTCCTCGCCATGAGGATCGCGACAGGCGCGTCGAGGGGGCAGGAACACCCTTGCGGATTGACATTCTTGGTGGTGTTTCGTTGTTGTCGGTAGTTGTTGTTGACGATGAGGATAAATAAAAATGACCTTGTTTTTGTTCTGTTTTCTCTTGTTGGGAATCGTCGACTTTGAATTCTTCGAGTTATCGGAAAGCTGAGGTACCCAAATGTCTGTAGCTTTTTTCTTTTTACCCTCTTGTTTATCATCTGCGATTCGTGGTAGGTAGGAGAGGGAAATGATAATCCGAGATTAAGGAAAGGAGAAGATAAAAAATAAAAAAAAAATAATAAAACAGAAGCCGACCGGCCGCCGACCCGTTCCCCAGGACCAGCCTACGAGGAATGGATAACGCGGTGGCGACGGCAGCGGTGGTGGCGCTGGGGGTGGCGGCAGTGGTACTGCTGATGGTAGTCGGGACGGAGGAGAGGCGATGCATACATACACGCGTGCATGCTGCATGGGTGGATGGTACGGCCGGGAGACGCGGAAGAGAAACTCACATAAAAAGGTGACAAAAAGAGCGGTTGAAAAAAGAAAACGAGATTCGACCAGACAGAAGAGAAGGACCGGGGCTTGGCGACCCTTCCACGACTGCTGTTGTCATCTCGGCTCCCCCGTCTTCTCCCGGCCACGGGCGGCTAAGTCACCGCCGTTCTCCCCATCCGTCCGAGCGCCGACCGACCAGCCGGCCGATTCGCCCGCCGGGGCTTCTGGAGAACGCCGGGGCAGCAGCGATCTGGGGAAGCCGCTAAACCCCTGCGTTTTTATATGGTAGCTCTGCCGAGCGCGGGCTGACGCGTTGAGTAAGCGGAAAGACGTGTGTGACGAAAAGGGGTCCCATGGTATTTCACGTGACGATGAGGAGATGCGGTTTGGAGCACATACGGTTTAGAAAAAGGGAGTTGTCGTGACAAGGGCTGAGGGACCTCTGTCTCCATGTGTGTATAAAAAGCAAGGCACGTTCATAATGTAAAAAAGAACACGTTGTAAACAAGCTATTGCTGTATCATTCGGCTGACTATGCTTCATTCGGACTGATTTTCTTTTCCTAACGGCGTAACTTAAAGTGATTAACGTATGATATTTGTTCCCCAGAGTTATACTATAGTCATCATCCTAAAATTCAGATATAAATGAACACATGTCGTATGGGATTATTAAGAAACCGAAACTCTCCACAGTTCACCATCTTCTTCGTCATTCAACCGATGACCCACTCCGTACAACGAATCAGTCTGCTGTGTCACACTGCAAACTACTAGCGACGTATGCAAACAACTTGAAACACGGGCTGTTGTATTGACGACCGTTGTACCATTACTAGTCACATTGCATAGAGACCATCCACCGTCATCCCATCTTTCCCACCCGATGGAAAACCGTCTTCTATCATCAACTATGGTAAGATTTCGACCCTGCGAGGTATTCAGTTTCCCCATATCCATAACCTGGATTTTATCATTAAACCCCAATATTAAACACTTTTTTAGTACCCCCCCACCCACCAAAAAATGTGACTGGACCGGTTCCTAGCAGCTCTGGGAGCCATGTTCAGGTTGAACCACAGCTACAGCGAAACCGAGTCCAGTGACCGGTAACCACGTCCAGCCCCTGCGTATGTACCAGTCCAAGCACGTCCGGTCATTGTTCTACACAGGAAATCTAACTAGGTCAACGCAATTTTATTCCACCGTTACGCAGAATACTAACAAACAAACACACAAATTTAACGAATTACACGTAGTTTATTACATGAAAACTGTAAGAACACCAATTCACTAAGCGATACAACATTTAGCTGACTTCCAAGTGCCACACATCACCACTGTATTCATCCATGTTTTCACCGAACCAACGAGACAGATCGAAGAAGCCAGAATCTCCCGACTTTAAATTACATAAATCCAACGTATTATGACCACAGCTCGACACACAAATAGTTGCGTTACTATTCACAGTAGCATTACCTATACCCGTAACGTTGCACAACCACTGATCACCATTGTTACCAAAAACGGTTTTCCACTTAGTTGTCAACGGATCTTTCCCATGCGTAATGGTCAAATTACTACCAGTCGTCGCTTTTAGCTCATTACGAGTATTATCCGCATCCACATATATCAACGTCATAGCTAGGCACGCTATAAGTACCCCCCCCCCACAATGGAATGTTGCCAAACCGGTTCTTTCCCGTTATAGCCATAGCGTTCCCAGGCAAAAGCAAACGCCAAACCTAATGCAGTGAAAAGCGCTTGCAGCCAGAACCAGCTTATGTACCAGCCACAATCACATCCGGTTATTGTTTCCACAGGAAATCCTACCAGGCAAAGCCCCGCTTGTTTTGTTCCTGACCATCTTGTTTAGCAATTCGTAAACTGTCAGCCTAGCGACGTCCGTTTAGATCAAAAGTCACGTATATAGCGACGCTGTTTCCACCCGTTTCCCCGTCCCGCCGTTTCCGAACAACCCACCCGGGTTCAGACAACCGACCACCAACAGAAATATACACACAGACCACCGGGAGTTCAGTTAAAGATTTCATCAGGTTTATTTTGGCTGCTGCTAGTCTTTTGCTTCTTAGAAAAAAAATACCCATATAGAGAAATAATGATAGTTTGACAACACATATGGCAGGGATTTCTTCTTCATCAATAAGATATGCAATTCCCCCAGGGAGAGACTTTCAACAATTGAATTTACAAAAACAAAATTACATCAGGAGAAAGAGAGGATACATTAATAAATATATTATATCTGGTGTATATACTGAATGCTGCTGGTTCATAAGGTAACGATGCTACTTTTTTTAATTCCAAGATGGTTTTTCTTTGTTAGTCTTTTGTTGACTTGCTGGTTCCTAAAAGTTCGCAAAAACGATTGTGTGAAGATTATGACGTTGGTTGACTAGTTCATGAGATTCTGCTGTACGTGTGATGGTTATTCGCTGGTTCGTTCTAAGATGAGTATCGTACTGTGTCTGCGATGGTCGTCTCTTACTGGCATTCTCTCGGCTGCCTCTTGTTTTCATGATTGAAAAGGAAAAAAGGACTCCGAGGGCGCGGTCATCTTTTACTTTTCGGTTTTCTCGTTGGCGGGTCAGAGGTAGTCAGATCATGAGACTGTCGTGGTCGATGAAACTGTGTCTGCTCAAGTGACGTCCATTTCTTGTACGGAGAAAAAAGTCATCGGGATAAATAAGGCTATACAAGGCGTTGTCAAGCGTGCGGCTCTAAACAAATTAAGCGATACAAAATTACAGTGATACGAATAATAAATTACCCCCTCCCCCTGTGGTCCCCCCGAGGCGAGAGCCACCCATCGTGTACTCTCGCACCACCCACGACCACAGGGGGAGACGGGACGAAGAGACGACGCAGAGCGCCATCTCCTCCTGGAGGCCGGCGGCGTTAACTGCTACAGCTGCGGCGGCGACGACAGCTGCGATTTGTCGGCCGACATGCCGATGGTATGGGCGGCGGCGGCGGTGGCCGCGGCAGCGGGGAGGAGAGGAGAGAGAAGAGGAGCGGGGCGTCCGAAGGCGAGGATGGCATGGTCTCGCCGGAGCGCCCGGCTTTTATGGAACACTCGCGTCCGGTTGGGTATCACCCACAGGAAGATGAATCACAACTTCCAAACCATCTTGAGACCCGAGTAACGGTTTACAGGTCGCACGCCAGTCTCAGCTAAAAACAGCGGACAGTCCCACGCTGTTTCTGTTGTGGCTCTCTCCAGTTTCCTCATCGCCGTCTTGGTCTCCGTCATCATCGGAAGAATACCACCCGCTCTCATGCGGCAGTCGATCAGCCTCGATGAACGAGACGCGGCGACGCCTTTCTACGGCCGACTGGTTGTGGTGGTGAAAGAAGAGCACCAGCAATCCCAGGAGGAGCAACAAGCCCTCACATGTCCAGGAGGTCGGGGAGAGGGCCTGTCGGAGATGACCGTGAGGCATCACGTACGGCAGCTGAGGAGAAACGGAGAAGAAAGGAAAATTACCGTCAGGGGCCGGGGTTCTTATTAGAGAAACAGCACGTAGGTCAGGATCCAGATGCTAATGGCAATCATGATGACGATGATCATGCAGGCCAAGACGCGGCGCACCAATGCAGAATCCAATAGCCGCCGTGCCTCCGGTTGGTGGCCGGCGGCATCTAGAGACATGATTTGGGGGGGGGACCGGCGGCGCAAAAAGACAGGGAGATGGACAGTGCCACGGTGTTTTGTTATGATTAGGACATGGGGACCGGAAGCCGAGACAGAGTACTACAGGGTGTTGAAGGGTAACGTGAGGGAGATCATGTCATGGGCGGGCTGAAGACCGTGCGGGGAGGATCGACGTGTGCGGTGCTTGTGGAACACGGTGTTTTAATATGTATCCGCGTGTAATGCACGCGGTGTGCTTTTTAGCACTCGGCTTGATAAGCTACGTGACCGTCTGCGCTGAAACCATGGTCGCCACCAACTGTCTCGTGAAAACAGAAAATACCCACCTAGCATGTAAGTGCAATCCGAATAGTACATCTACCAATGGCAGCAAGTGCCACGCGATGTGCAAATGCCGGGTCACAGAACCCATTACCATGCTAGGCGCATACTCGGCCTGGGGCGCGGGCTCGTTCGTGGCCACGCTGATAGTCCTGCTGGTGGTCTTCTTCGTAATTTACGCGCGCGAGGAGGAGAAAAACAACACGGGCACCGAGGTAGATCAATGTCTGGCCTATCGGAGCCTGACACGCAAAAAGCTGGAACAACACGCGGCTAAAAAGCAGAACATCTACGAACGGATTCCATACCGACCCTCCAGACAGAAAGATAACTCCCCGTTGATCGAACCGACGGGCACAGACGACGAAGAGGACGAGGACGACGACGTTTAACGAGGAAGACGAGAACGTGTTTTGCACCATGCAGACCTACAGCAACTCCCTCACGCTTGTCATAGTCACGTCGCTGTTTTTATTCACAGCTCAGGGAAGTTTATCGAATGCCGTCGAACCAATCAAAAAACCCCTAAAGCTCGCCAACTACCGCGCCACTTGCGAAAACCGTACACGCACGCTGGTTACCAGGCTTAACACTAGCCATCACAGCGTAGTCTGGCAACGTTATGATATCTACAGCAGATACATGCGTCGTATGCCGCCACTTTGCATCATTACAGACGCCTATAAAGAAACCACGCGTCAGGGTGGCGCAACTTTCACGTGCACGCGCCAAAATCTCACGCTGTACAATCTTACGGTTAAAGATACGGGAGTCTACCTTCTACAGGATCAGTATACCGGCGATGTCGAAGCTTTCTACCTCATCATCCACCCACGCAGCTTCTGCCGAGCCTTGGAAACGCGTCGATGCTTTTATCCGGGACCAGGCAGAGTCGGTGTGGTCACGGATTCCCAAGAGGCAGACCGAGCAATTATCTCGGATTTAAAACGCCAGTGGTCCGGCCTCTCACTCCATTGCGCCTGGGTTTCGGGACTGATGATCTTTGTTGGCGCACTGGTCATCTGCTTTCTGCGATCGCAACGAATCGGAGAACAGGACGTTGAACATCTGCGGACGGACCTGGATACGGAACCTTTGTTGTTGACGGTGGACGGGAATTTGGAATAAAAGATGCGTAACACCTGTCGAAGATGCGATAACTTTACATACAGGCAAACAGTGTATACAATTATAGTATTTTGTATGTTGCATAAAGTTACATGCAACAGTACTGCTAACAGTACTGCATCCATTACGCTATCCAACACTGCCTCTACCACTTTTGTAACCAACATATATTCAACTCCGAATAACAACACATCAACGACGCCACACACATCTGTCACCTCACAAGCGTCAACCATTGGCAACATCACCAACGTTACCTCCGACTTGAGTACTTTCACAACCGTATATTCTACATTCAATACATCATTTGCCAATATATCTAATACGGCTGTCACTACAGAATTGATTTCAACAAATACCAACACTATCTCATCTTTTACCAACGTAACAGCAAACGCTACATCATCTTATAACACAACAATCACCGTAACTGTCACGTCAGATGAAACTTCGCACAACGTATCCACTAATAATGCACTTATAAGCACACCATGGCCTACAAATTGCAGCGCCACAACATACACCACGTACAACCTTACTAACTCTTCCAACGCTTGTCACACAGAGACAACAATCATACGTTTCAAGGAAACCAATACAACAGGAATAGAAGGGAGTAATGTCACCATAAAGGGTAATTCTACGTGGGACTGTCTTTCAGTCGCCTGGATACGACATTACAATAGATCCACACACGGACATCATCTAGGTTATCGTAAGAACGCACATACCCAATCTTGGTATTGGCTACGCATCCTTACCTCTCACACTGTATGTCATTCTCAACATGAAAGACCTTCACTGTACCATGACTTATGTCGTTCGTGCAACAACACAGAATTACATCTGTACGATCTAAATATCACCAATTCCGGCAGGTACAGCAGACGTTGTTTTAAAGAAAATTACTTCACAGGACATCACGAAGATGAAAATTTCTACCTATTAGTAACACCAAAAAATCATACTGAAGCTATTAATGCTACTTTCGTTTGCCCTAGATACAACACCGATATCGAAAATGAAGATAGAGAGAAAGGAAGTCAACATACTAACAATACACATCACCACAAACGTAATCTCTATCATAGCTCGCAAAGAAGCCGCACCGTATGGACCATCGTGTTGGTTTGTATGGCCTGCATAGTTCTGTTTTTTGCACGACGAGCCTTTAACAAAAAGTATCATATGTTACAAGACACCGTCAGTGAATCAGAATTCATTGTTCGATATCACCCAGAACATGAAGATTGAGCTACGTTTCCGGGCAGACATCTTATGAAGCTGAACAATAAACTAAAACATTCTGTAAGACTCAGCGTTCAAAGGAATATTAATGCCCATTGAGCGAAAACTAATATTGCAATGGACTGGCGATTTACGGTTACGTGGACCGTTACTTGTGATGGTTTCAATTATACAGTCCATAAAAGATGCGATCGCAGTTACGAGGTAATCAACGTAACAGGATACGTTGGTAGCAACATAACTCTAAAAAAATGCAATCAGACTGAGAAATGGCACAATGTAGACTGGATTCATTATGAGTACCCCACGCATAAAATGTGCGAATTAGGCAACTATCACCAAACCACACCACGGCACGACATATGTTTTGACTGCAACGACACCTCCCTAACTATCTACAACTTAACCACAAAAAACGCTGGAAAATATACCAGGCGTCACCGTGATAACGGTCAAGAAGAAAATTACTACGTAACGGTGTTAATTGGAGACACAACGTTATTCACTCTTGGCACATGCCCTGTAAGATATAAAGAATCTACGAACACTGAAAACACCATTGGAAGTAGCATCATAGAAACCATTGAGAAAGCTAACATTCCCCTGGGAATTCATGCTGTATGGGCAGGCGTAGTGGTATCAGTGGCGCTTATAGCGTTGTACATGGGTAGCCATCGCATTCCCAAAAAGCCGCATTACACCAAACTTCCCAAATATGATCCAGATGAATTTTGGACTAAGGCTTAACATGCTGATCAATAAACTTTTTTTAACCAATAACATGTCTCCGTTTTTTTTTGTTAACAACCTATGATATAAAGCGTTATATTCAGTCGTTACTAAACAAAAAAACATGGGCATGCAATGCAACACTAAATTGTTATTGCCAGTCGCACTAATACCGGTTGCAATCATCCTAATTGGTACTCTAGTGCCGATACTTTTACATGAACAAAAAAAGGCGTTTTACTGGCGACTTTTTCTGCAAAGTCAACATGTAGAAGCACCCATTACAGTAACGCAGGGAGACACAGTCTACCTAGACGCTAGCAATAATCCCTGTAATTATTCCAGCTTTTGGTACCACGGTAATTGCGAACTTTGTGGATGGAACGGATATCTACGCAATGTTACACATTACTACACAAACACATCGTGTTCCCCGCAATTCATCTGCATAAACGAAACTAAAGGTCTGCAGTTATATAATGTAACATTAAACGATTCAGGCGCTTATACTGAACACGTTTACGAATGTGACCTTTCGTGTAACATTACTACTAATAACGAATATGAAATACTCAATTATTTTGATAACTGTAACTACACCATAAATAGCACCAAGCATATTATCACCGTGGTGTCTTCACGTCATTCTAAACAAACAAATTCCCACGTATCCACTCACGCTGGTTGGGCAGTCGCCGTGGTGACGGTAATTATGATCTACGTTCTGATCCACTTTAACGTCCCGGCAACTCTGAGACACAAACTACGAACTAGAAACAACGTAAATCGCATAGCGTGATTATAAAGTATCGACGCTAATTTCTCCAAGATAAAATTTGATTACTCCGTGCAGTTCTCAAAAACTGTAAGGCCCCGCTTTTCCACTCCGTCATGAAGGATCGCAATAGAATACTGCTATGTATCATCTTTATTTGCATTATGTGCCTCATTTGTATTTACTTTAAACGTCGTTGTGTTTTTACTCCGTCTCCAGACAAAGCAGATCTGCGAGTGGAATTTCCCTCGTTACCCCCGTGTATTGGCATACAGTGCGCTGCATGAGAACACGCGTGACACATAGCGTACCCCTGGACGGTACAGTTTATGATAACGTAATTCAGGGAAAGTATACATTCATACCAACATGTTATCACATAACACACAGATTTTCTGCGTGTTTTATAAAAGAGCGTCTCGAAGCAGCTTGAGCCACACTACGGTCCAGATGACGAGCGTAATTAAAAATATGCCGCGCAGTATTCGAAAGCCGTACTGAGCGTGCGAGGCGGGTAGGGTGCCGAACGACGGATATGCGTCGTTGTCATCTTCGACTATAAGGATCGCGACCGAGTCTTCGGCCATGGTAAACGTCACCCTGTGTGGCTGGTATGTAGCGTATCCGGTTTGGAATTGTTCTGCTCCAGCTCGGGGGATAGTGAGGAATTCTCAAGGGATACGGGACCCAATGACTGGATAAGAGAAGGGTTTTTCCCCGTAAGATGATCCTCGTATCACATGAGGTCTGGATATGTATAAATGAAGAGTGAAATAGGCACAGGGAATCAGATGCCAGCCTCGTGATGCAGCCGCTGGTTCTCTCGGCGAAGAAACTGTCGTCTTTGCTGACTTGCAAATACATCCCGCCTTAAGCGATGAGTCTATAAAGCACCGTTGCCCGAGTACGGTAAAAGTGACCCGGATTGTAGAACGTCCTTTTTTTTTGTTTTTGCATCGTTTATCGTCACTACTAGTGCAATATTTTGATTGTAAGGCTGAAAGAGTATCGTTATGATGCTTAGAACGTGGAGATTATTACAGATGGTACTGCTTGCCGCGTACTGTTATTATGTTTTTGCGACTTGTTCAATCAGCACGACGACTGCTCCTGTGGAATGGAAGTCTCCCGACCGTCAGATTCCCAAGAATATTACCTGCGCTAATTACTCAGGGACCGTCAACGGCAACGTTACATTTCGAGGTCTTCAGAACAAAACGGAAGACTTTTTGTACTGGTTGTTAGGATGGGGTCATAAGTCCATTTGTTCGTTCTTCCCGAAACTCCAGGGTAACTATGACGAACAACATTACAGATATGAAGTAGCGAACCTGACGTATAACTGCACCTATAACCGCTTGACGTTGCTGAATCTGACGACGGAAAACAGCGGAAAGTACTATTTCAAAAGGGAAGATGCGAATTTCACCTTCTATTACTCTTGTTACAACTTGACCGTGTCCTAAAGATCGCACGTGAAGTTTCACAGAGCCGCGTGGCTGTAGCTATTGTGTTTACGTTGCTTTTGAAATGTTAAGCGTCCCTACGGCGCTAACATGTTTCTAGGCTACTCTGACTGTGTAGATCCCGGCCTTGCTGTGTATCGTGTATCTAGATCACGCTTAAAGCTCATGTTGTCTTTTGTGTGGTTGGTCGGTTTGCGTTTCTATGATTGTGCCGCGTTCGAGTCCTGCTGTTACGACATCACCGAGGCGGAGAGTAACAAGGCTATATCAAGGGACGAAGCAGCATTCACCTCCAGCGTGAGCACCCGTACACCGTCCCTGGCGATCGCGCCTCCTCCTGACCGATCGATGCTGTTGTCGCGAGAGGAAGAACTCGTTCCGTGGAGTCGTCTCATCATCACTAAGCAGTTCTACGGAGGCCTGATTTTCCACACCACCTGGGTCACCGGCTTCGTCCTGCTAGGACTCTTGACGCTTTTCGCCAGCCTGTTTCGCGTACCGCAATCCATCTGTCGTTTCTGCATAGACCGTCTCCGGGACATCGCCCGTCCTCTGAAATACCGCTATCAACGTCTTGTCGCTACCGTGTAGCTAGTTAGCCAGCTGTGTGTAGTGTTTTGCTTTTGCATATTTGTTTTCAGTCAGAGAGTCTGAAACGGGGTGGGAGGGACTTTTGCGGGTAGTGCATGCTAAGATGAACGGGTGGGCTGGGGTGTGCTTGATAACTCACTGTTTGAATACGCGCTCACGCACATATGTAGCACTCAACATGTTAGCTTTTGCCCGCACGCCCCGGGGCGTGCCGAGCTGCCTTTTTAATAAAGTCTGGGTTTCCAGATACGCGCTGGTTCTGATTTTGATGGTTTGTGCCTCTGAAAGCTCTACGAGCTGGGCCGTGACATCCAATGGACTGCCTAACTGTAGCACGGTAACTAGAACAGCGGGTCAAGACGCTGAATTGCACGGTCCGGCACCGTTAAGCTGTAATGTGACCCAGTGGGGACGTTACGAGAATGGAAGCACACCCGTGTTATGGTGCACTTTACGGGGATCAAGCATGCGAGTCTCATTAGGACACCGTGTAGCGTTTGGCTGTTCTTGGAAAACATTTTTTATTTATAACGTTTCTGAAAGTAGCGGTGGCACTTACTATCAAAAAGGTTACAACTGCACCGACAAACATATAACACTATCTTGTTTCAACTTAACGGTGGTTCCTCGAGCGGTTCAAAGCACAACCACCGTAATGACACCCACGCTGGTTACAAACTCCACATTCAGTGTGTCACTTGTTCCGTTGAGACTGACGACAAATTCCAGCGCGTTTGGACACGCTATTTATCAACGACAACAGCGTGTTGAAAACGGGACGTTATCCAAGAACATAACTAACTTGGCATTCACCTATGGCAGCTGGGGCGTTGCGATGCTGCTGTTTGCCGCCGTGATGGTGCTCGTTGATTTGGGTTTGCCTCAATCGGCTTGGCGACGCTGGCGAAGCCACGTGGACGATGAAGAACGTGGTTTGTTAATGTAGGAAATAAAAGGCAGTTTGAGCATGACTGTTTCCAAACCGTAACGTGGTAAATAAATCATGGCTTCCGACGTGGGTTCTCATCCTCTGACGGTTACACGATTTCGCTGCAGAGTGCATTATGTGTACAATAAACTGTTGATTTTAACTTTGTTTGCCCCCGTGATTCTGGAATCCGTCATCTACGTGTCCGGGCCACAGGGAGGGAACGTTACCCTGGTATCCAACTTCACTTCAAACATCAGCGCACGGTGGTTCCGCTGGGACGGCAACGATAGCCATCTCATTTGCTTTTACAAACGTGGAGAGGGTCTTTCTACGCCCTATGTGGGTTTAAGCCTAAGTTGTGCGGCTAACCAAATCACCATCTTCAACCTCACGTTGAACGACTCCGGTCGTTACGGAGCAGAAGGTTTTACGAGAAGCGGCGAAAATGAAACGTTCCTGTGGTATAATTTGACCGTGAAACCCAAACCTTTGGAAACTACTCCAGCTAGTAACGTAACAACCATCGTCACGACGACATCGACGATGATCGACGCGAAAAGTAACGTTACAGGGAACGCCAGTTTAGCACCACAATTACGTGCCGTCGCTGGATTCTCCAATCAGACGCCTTTGGAAAACAACACGCACCTGGCCTTGGTAGGTGTTGTTGTGTTTTTAGTTCTGATAGTTGTTTGCATTATGGGGTGGTGGAAATTGTTGTGTGGTAAACCAGAGTTATAGTAATGTGCTTTTTATCAGGGAGAAGGTTTTGTGCCAACAATGACTAGCCCGGGACTATCTGCGTCAGAAAATTATGACGGAAATTATGAATTCACGGAAACCGCCAATACAACGCGTACAAATAGAAGTGACTGGACAACGTTAGAAACCAGTGCATTGCTATTGAAAAACACGGAGACTGCAGTGAACCTCAGCAACGCGACTACGGTCATCCCACAACCTGTAGAATACCCGGCTGGGGAAGTACAATATCAAAGAACGGCAACGCATTATTCTTGGATGCTAATCATTGTCATCATTCTCATCATTTTTATTATCATCTGTCTACGAGCACCTCGAAAAATCTACCATCACTGGAAAGACAGTAAACAGTACGGACAAGTGTTTATGACAGACACGGAACTGTGACAGTGATGTCTAAGCGTTTGCAGGTATTTCCATGGATAACAATTTTATTTTACACATCAAAATCCCAGTATTGGAACTATATGGCAATACCATGTACCCCTACAGTTGGATACGGCAGTCATAATATTAGCTTGCATCCGCTTAATAACTCATTATTTCAAGACGATGTTTTTGAATGGTACATAGACAAACCAATGGTTACAAGTTATGTCTTTATCAAAGTAATGAACGCACAAAATCCAATCTAGACTCTCCAAATATTGTGTGGCAATGCACAGATAATCGTACACTAATTCTCATGAACTTAACCACAACATACAGTAGAAACTATTATTTTCAATCCTTTAAATATCTCGGACGAGGAGTACCAAAACCGAATAACTTGTGTTATAACGTTAGTGTACACTTTACCCACCAAACACATTGCCATACAACTACATCATCCCTGTATCCACCTACATCTGTACACGATTCATTAGAAATATCACAGTCATTCACCTCAACCAACTTCACACATACCGCGGTCCACTACGCCACCGGTAACGTTGAAGCACAACACGACACTACCACTCCACATACAATGTGGATCATACCCCTAGTTATCGTTATAACAATCATCGTTTTAACTTGTTTCAAATTCCCCCAGAAAGCTTGGAATAAATTCACACAATACAGATACAGCGGTATGCTCGCCGCCGCTTAAAGAATCAACGCCAAGGAAACCAAAACGTAAAAAGAATAGATATGTACGTTTATTTTTCAGCTCACTGTTTGAATACCGTAAACATAATGACGTACATATACGTGGTTATACAACAGGTGTTTGTGTTATGCGGCGACTGATTAACCATATCGTGAACCATGATCTTTTCCGATGGTCCGTCGTGACCGCAATGATATTTTACAGATATTCCGAAACCTGTATGGAGGTCACTGTCAGAGTAGGTGATCCAGTTACCCTCGGTAGTGGACATGGTTATCATCCAGGTAGGGATAACAGGGTAATGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGG SEQ ID NO: 11 DNA Genus/species-Cytomegalovirus HCMVDescriptive title-HCMV fragment post-editingACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGATTACCCTGTTATCCCTACCATTCCGGGCCGTGTGCTGGGTCCCCGAGGGGCGGGGGGGTGTTTTTAGCGGGGGGGTGAAATTTGGAGTCTTGGAGCCGCGTGTGCTGTGGAGGACGGTGACGGTGGTAAGAGTGTGCTGCGGTGCGGTTGGGACGGCGGCGGCGAATAAAAGCGGCGTGCGGCGCGCACGGCGAAAAGCAGACGCGCGTCTGTGTTGTGTGTCTTTGACCGCGGCGGAACACACGCGGAAAAGCGAGTCCCAGGGGACACACGACGAGCGAGTCCCAGGGGGGGACGACGACGGCCAGGGACGCGGAAACGACGCGGAAAAGAGGAAGTCCCCAGGGGGACGGGCGGAAAAGAGGAAGCGCCTAGGGGACCGCGGGGGCAGGAACAGACGAAGTACGCCGCAACCCGCGTCGAGGACACACGCAGAAGCGGCCGCCCAGGGGAGGGGGGGGGGGGGACTCGCGGGCCCCGGGGCACACTTGTTGTTCCCTCCGGCCGCCGACACGCACCCCGAAGCCGCGCACACCGCCGACACACCCCTGACACACCCGCGACACACCCGCCACACGCCCGACACACGCCCGCGACACACCCGACCGACACACCCTGACACACCCCGCCAACACACCCAGCCGCACCCGCCCCGCCAACACACCCCCGACACACCCGACACACGCCCGCGACACACCCGGCACACACCCACCCACCCAGCCGCGCCCCCGACACACCCCGAACGGCGCCGGTGCGGGACAGGGCTCACGGAGGTTTGCGGGCCGTGAGCACGCCTCCCTTTGTACACACTACCGGTGCGTGGCGTCCCACGCTATTTGTTCGCGAGACCGGACTAAGGGAGGTTTGCGGTGCGTCAGCGCGGGGCGGCGTTTGCGGCGTGTTTCGACCAGCGCTTTGTGCGCGCTGCCTGTGCGTGTCGTCCCATGGTCTTTGTCAGCGGCACGGCGCTGGGGACGGGGTTTCACCGCGCTGAGGGATCTTTCTGCGGGTGTGAGGGACGGAGCTTTTTTCGCACGCTGGGCACCGGGCTGGGGGACGGGGGGTGTGCGGGACGGCGGTGGGGCCGGGGCGTTGCGGGTACGGGGATTACGCTGGGAACGGGGACTCGCGGACCCGGGCTGAGGGACGGGGGTGGCGGGGGTGTTTGCGGCGAGGACGGGGGCCTTTTGCGGCGGGGACGGGGACTCACCCTCGCCTATTTAACCTCCACCCACTTCAACACACACATGCCGCACAATCATGCCAGCCACAGACACAAACAGCACCCACACCACGCCGCTTCACCCAGAGTACCAACACACGTTACCCTTACACCACAGCAACACACAACCGCCTATCCAAACCTCGGACAAACACGCCAACGAAGAACACCGCACGCAGATGGAGCTCGACGCCGCGGATTACGCTGCTTGCGCGCAGGCCCGCCAACACCTCTACGCTCAAACACAACCCCAACTACACGCATACCCCAACGCCAACCCTCAGGAAAGCGCTCATTTTTCCACAGAAAATCAACATCAACTCACGCATCTACTTCACAACATTGGCGAAGGCGCAGCGCTCGGCTACCCCGTCCCCCGCGCGGAAATCCGCCGCGGCGGTGGCGACTGGGCCGACAGCGCGAGCGACTTCGACGCCGACTGCTGGTGCATGTGGGGACGCTTCGGAACCATGGGCCGCCAACCTATCGTGACCTTACTGTTGGCGCGCCAACGCGACGGCCTCGCTGACTGGAACGTCGTACGCTGCCGCGGCACAGGCTTTCGCGCACACGATTCCGAGGACGGCGTCTCTGTCTGGCGTCAGCACTTGGTTTTTTTACTCGGAGGCCACGGCCGCCGTGTACAGTTAGAACGTCCATCCGCGGGAGAAGCCCAAGCTCGAGGCCTATTGCCACGCATCCGGATCACCCCCATCTCCACATCTCCACGCCCAAAACCACCCCAGCCCACCATATCCACCGCATCGCACCCACATGCTACGACTCGCCCACATCACACGCTCTTTCCTATCCCTTCTACACCCTCAGCCACGGTTCACAATCCCCGAAACTACGCCGTCCAACTTCACGCCGAAACGACCCGCACATGGCGCTGGGCACGACGCGGTGAACGTGGCGCGTGGATGCCGGCCGAGACATTTACATGTCCCAAGGATAAACGTCCCTGGTAGACGGGGTAGGGGGATCTACCAGCCCAGGGATCGCGTATTTCGCCGCCACGCTGCTTCACCGATATCCAATAAACCCATCCCCTCGCCACGACGTCTCCGCGTATCTTTGTAGCCTCAGGAATCCGTCCCCACGTCCATCCATCCCGAGCACTCCACACGCTATAACAGACCACGGACACGGCAAATGCATGCAAACTTCTCATTTATTGTGTCTACTACTCTGTGTTGCTACAGGGAGTGAAGGGGGTGAAGGCAAAGAAAAAAAAAAGGAACAAAATAATAGATTAGCAGAAGGAATAATCCGTGCGACCGAGCTTGTGCTTCTTTTCTTATAAGGAGGCAAATATACTAGGGAAAACTTAAGAATAGGAAGAAACCGAGGTTTGGGAGAAAAGCTGAGATAAAATAGCGCATTTTCCATACAGAGGTTGTTGTTTTTGTGGATCCTAAGAGGTTTCAAGTGCGAATCTCAAAGTTCTCACGAGAATATTGTCTTCAAGAATCGACAACTGTGGTCCAAGATTTTTTTTTGGTCTTTTTAGGTTCTGCGAGGGACATCACGATGGATCGTTGCGATGAAGTCACGCGTACGCCTCTGGTGTGGCGCGGTGTCGTGACAGGAGAGTGTGTTTTCAGTGCAGAGCTGTCTTGATTCCTATATCCGAGTATCTGTTTTCTCGTAAGGACGGTAATCTTCTTTGGTGTAAGTACATCTAAAAGCTGCAAACTATATTTTAAGGGCTGTCTCTAGGTGTACTTTGATGCTGGAGTTTTTCGCTGTGTTGATGTGAATAAATCTACTACTACTATTATATGCAGAAAGAGTGATTATGCCGAGACAAGATTGCATTGGCTGAACTGTTTCAAAAACGCCTACACTCTACTTATCCGTAAACCTAAGGTAATACTATGTGTAAGTTGTTTTTTTTTCTTTTTGTAGTAAAATGGTGATACGTGCAATTAAAACTGTATTCCATGTTTCCATCCTTTCATTTCAACTTTAAAGGCGGCTTTGAGAGCGAAGAAGTGCGAGGATAAAAATGGATGACTCCTTCGTGTCCAGGGAGTCGACTACTGCAACGCTGATTGATTAAAAGATGGTCTCCGATGATGATGTTGTTATTGATCGAATCATGGTGCAGAACGGCGACGGAGAGGAGCGTGTCCGCCGCCGGGAAGGTGGTCTCTTTCTCTTTTCTTTTTTCAAGAAATCTTCCATGTGTTTATCGTAGTGATCGAAATCGACTGATCTCGGGTTCTTTTTGTTGGTTTCTTTTCGGTTAATCATGTATTGTTTTCTTTTTTTACAGAAAGATACTTTTTTCATGAGCAATTCCTCGCCCGGCGCCGGCATGCCGAGGTGGGGCCACTGCGATCAGCGGCATGCCGACGCCGACCCGGGGATCTTGGATTCACCGTTTTCTCTCTTCTCTCTCTACATACAGACCGGGTGGCAGGAGCGGTAAGGAATCATCGTCGTCTTTCATTCTTCGATGATTATGGTAATACTAAATCTTATCTAGGAGCATATACATCTAAGATTGGAGTACTAGTAGTCGTTTGTGGTTTCTATTTTTTTTATATTTATCTATGACAGTTTTTCTGTTTTTCGTTTTGATAATAATATAATAAAAACTCATGGACGTGAAATCTGGCTTGGTTGTGGTGATTTCATTCTCATTATTGTTGTTTTCTTTCCGTCTTGCGGATGAAGATGTTGCGATGCGGTTGTTGTTGGTGTTGCTATACACCGAGAGAGATGATCTTTTTGTTCTTCTGGTTCATTTCCTATGATTGTTTGGCTGCTGACCGACGCGTCAGGATGTGCAGGGCATGCGGGGAATCAGGACCGGACACGGGATAATTTCATCTACCTATACGGAGATCGCGGTCCTCGCCATGAGGATCGCGACAGGCGCGTCGAGGGGGCAGGAACACCCTTGCGGATTGACATTCTTGGTGGTGTTTCGTTGTTGTCGGTAGTTGTTGTTGACGATGAGGATAAATAAAAATGACCTTGTTTTTGTTCTGTTTTCTCTTGTTGGGAATCGTCGACTTTGAATTCTTCGAGTTATCGGAAAGCTGAGGTACCCAAATGTCTGTAGCTTTTTTCTTTTTACCCTCTTGTTTATCATCTGCGATTCGTGGTAGGTAGGAGAGGGAAATGATAATCCGAGATTAAGGAAAGGAGAAGATAAAAAATAAAAAAAAAATAATAAAACAGAAGCCGACCGGCCGCCGACCCGTTCCCCAGGACCAGCCTACGAGGAATGGATAACGCGGTGGCGACGGCAGCGGTGGTGGCGCTGGGGGTGGCGGCAGTGGTACTGCTGATGGTAGTCGGGACGGAGGAGAGGCGATGCATACATACACGCGTGCATGCTGCATGGGTGGATGGTACGGCCGGGAGACGCGGAAGAGAAACTCACATAAAAAGGTGACAAAAAGAGCGGTTGAAAAAAGAAAACGAGATTCGACCAGACAGAAGAGAAGGACCGGGGCTTGGCGACCCTTCCACGACTGCTGTTGTCATCTCGGCTCCCCCGTCTTCTCCCGGCCACGGGCGGCTAAGTCACCGCCGTTCTCCCCATCCGTCCGAGCGCCGACCGACCAGCCGGCCGATTCGCCCGCCGGGGCTTCTGGAGAACGCCGGGGCAGCAGCGATCTGGGGAAGCCGCTAAACCCCTGCGTTTTTATATGGTAGCTCTGCCGAGCGCGGGCTGACGCGTTGAGTAAGCGGAAAGACGTGTGTGACGAAAAGGGGTCCCATGGTATTTCACGTGACGATGAGGAGATGCGGTTTGGAGCACATACGGTTTAGAAAAAGGGAGTTGTCGTGACAAGGGCTGAGGGACCTCTGTCTCCATGTGTGTATAAAAAGCAAGGCACGTTCATAATGTAAAAAAGAACACGTTGTAAACAAGCTATTGCTGTATCATTCGGCTGACTATGCTTCATTCGGACTGATTTTCTTTTCCTAACGGCGTAACTTAAAGTGATTAACGTATGATATTTGTTCCCCAGAGTTATACTATAGTCATCATCCTAAAATTCAGATATAAATGAACACATGTCGTATGGGATTATTAAGAAACCGAAACTCTCCACAGTTCACCATCTTCTTCGTCATTCAACCGATGACCCACTCCGTACAACGAATCAGTCTGCTGTGTCACACTGCAAACTACTAGCGACGTATGCAAACAACTTGAAACACGGGCTGTTGTATTGACGACCGTTGTACCATTACTAGTCACATTGCATAGAGACCATCCACCGTCATCCCATCTTTCCCACCCGATGGAAAACCGTCTTCTATCATCAACTATGGTAAGATTTCGACCCTGCGAGGTATTCAGTTTCCCCATATCCATAACCTGGATTTTATCATTAAACCCCAATATTAAACACTTTTTTAGTACCCCCCCACCCACCAAAAAATGTGACTGGACCGGTTCCTAGCAGCTCTGGGAGCCATGTTCAGGTTGAACCACAGCTACAGCGAAACCGAGTCCAGTGACCGGTAACCACGTCCAGCCCCTGCGTATGTACCAGTCCAAGCACGTCCGGTCATTGTTCTACACAGGAAATCTAACTAGGTCAACGCAATTTTATTCCACCGTTACGCAGAATACTAACAAACAAACACACAAATTTAACGAATTACACGTAGTTTATTACATGAAAACTGTAAGAACACCAATTCACTAAGCGATACAACATTTAGCTGACTTCCAAGTGCCACACATCACCACTGTATTCATCCATGTTTTCACCGAACCAACGAGACAGATCGAAGAAGCCAGAATCTCCCGACTTTAAATTACATAAATCCAACGTATTATGACCACAGCTCGACACACAAATAGTTGCGTTACTATTCACAGTAGCATTACCTATACCCGTAACGTTGCACAACCACTGATCACCATTGTTACCAAAAACGGTTTTCCACTTAGTTGTCAACGGATCTTTCCCATGCGTAATGGTCAAATTACTACCAGTCGTCGCTTTTAGCTCATTACGAGTATTATCCGCATCCACATATATCAACGTCATAGCTAGGCACGCTATAAGTACCCCCCCCCCACAATGGAATGTTGCCAAACCGGTTCTTTCCCGTTATAGCCATAGCGTTCCCAGGCAAAAGCAAACGCCAAACCTAATGCAGTGAAAAGCGCTTGCAGCCAGAACCAGCTTATGTACCAGCCACAATCACATCCGGTTATTGTTTCCACAGGAAATCCTACCAGGCAAAGCCCCGCTTGTTTTGTTCCTGACCATCTTGTTTAGCAATTCGTAAACTGTCAGCCTAGCGACGTCCGTTTAGATCAAAAGTCACGTATATAGCGACGCTGTTTCCACCCGTTTCCCCGTCCCGCCGTTTCCGAACAACCCACCCGGGTTCAGACAACCGACCACCAACAGAAATATACACACAGACCACCGGGAGTTCAGTTAAAGATTTCATCAGGTTTATTTTGGCTGCTGCTAGTCTTTTGCTTCTTAGAAAAAAAATACCCATATAGAGAAATAATGATAGTTTGACAACACATATGGCAGGGATTTCTTCTTCATCAATAAGATATGCAATTCCCCCAGGGAGAGACTTTCAACAATTGAATTTACAAAAACAAAATTACATCAGGAGAAAGAGAGGATACATTAATAAATATATTATATCTGGTGTATATACTGAATGCTGCTGGTTCATAAGGTAACGATGCTACTTTTTTTAATTCCAAGATGGTTTTTCTTTGTTAGTCTTTTGTTGACTTGCTGGTTCCTAAAAGTTCGCAAAAACGATTGTGTGAAGATTATGACGTTGGTTGACTAGTTCATGAGATTCTGCTGTACGTGTGATGGTTATTCGCTGGTTCGTTCTAAGATGAGTATCGTACTGTGTCTGCGATGGTCGTCTCTTACTGGCATTCTCTCGGCTGCCTCTTGTTTTCATGATTGAAAAGGAAAAAAGGACTCCGAGGGCGCGGTCATCTTTTACTTTTCGGTTTTCTCGTTGGCGGGTCAGAGGTAGTCAGATCATGAGACTGTCGTGGTCGATGAAACTGTGTCTGCTCAAGTGACGTCCATTTCTTGTACGGAGAAAAAAGTCATCGGGATAAATAAGGCTATACAAGGCGTTGTCAAGCGTGCGGCTCTAAACAAATTAAGCGATACAAAATTACAGTGATACGAATAATAAATTACCCCCTCCCCCTGTGGTCCCCCCGAGGCGAGAGCCACCCATCGTGTACTCTCGCACCACCCACGACCACAGGGGGAGACGGGACGAAGAGACGACGCAGAGCGCCATCTCCTCCTGGAGGCCGGCGGCGTTAACTGCTACAGCTGCGGCGGCGACGACAGCTGCGATTTGTCGGCCGACATGCCGATGGTATGGGCGGCGGCGGCGGTGGCCGCGGCAGCGGGGAGGAGAGGAGAGAGAAGAGGAGCGGGGCGTCCGAAGGCGAGGATGGCATGGTCTCGCCGGAGCGCCCGGCTTTTATGGAACACTCGCGTCCGGTTGGGTATCACCCACAGGAAGATGAATCACAACTTCCAAACCATCTTGAGACCCGAGTAACGGTTTACAGGTCGCACGCCAGTCTCAGCTAAAAACAGCGGACAGTCCCACGCTGTTTCTGTTGTGGCTCTCTCCAGTTTCCTCATCGCCGTCTTGGTCTCCGTCATCATCGGAAGAATACCACCCGCTCTCATGCGGCAGTCGATCAGCCTCGATGAACGAGACGCGGCGACGCCTTTCTACGGCCGACTGGTTGTGGTGGTGAAAGAAGAGCACCAGCAATCCCAGGAGGAGCAACAAGCCCTCACATGTCCAGGAGGTCGGGGAGAGGGCCTGTCGGAGATGACCGTGAGGCATCACGTACGGCAGCTGAGGAGAAACGGAGAAGAAAGGAAAATTACCGTCAGGGGCCGGGGTTCTTATTAGAGAAACAGCACGTAGGTCAGGATCCAGATGCTAATGGCAATCATGATGACGATGATCATGCAGGCCAAGACGCGGCGCACCAATGCAGAATCCAATAGCCGCCGTGCCTCCGGTTGGTGGCCGGCGGCATCTAGAGACATGATTTGGGGGGGGGACCGGCGGCGCAAAAAGACAGGGAGATGGACAGTGCCACGGTGTTTTGTTATGATTAGGACATGGGGACCGGAAGCCGAGACAGAGTACTACAGGGTGTTGAAGGGTAACGTGAGGGAGATCATGTCATGGGCGGGCTGAAGACCGTGCGGGGAGGATCGACGTGTGCGGTGCTTGTGGAACACGGTGTTTTAATATGTATCCGCGTGTAATGCACGCGGTGTGCTTTTTAGCACTCGGCTTGATAAGCTACGTGACCGTCTGCGCTGAAACCATGGTCGCCACCAACTGTCTCGTGAAAACAGAAAATACCCACCTAGCATGTAAGTGCAATCCGAATAGTACATCTACCAATGGCAGCAAGTGCCACGCGATGTGCAAATGCCGGGTCACAGAACCCATTACCATGCTAGGCGCATACTCGGCCTGGGGCGCGGGCTCGTTCGTGGCCACGCTGATAGTCCTGCTGGTGGTCTTCTTCGTAATTTACGCGCGCGAGGAGGAGAAAAACAACACGGGCACCGAGGTAGATCAATGTCTGGCCTATCGGAGCCTGACACGCAAAAAGCTGGAACAACACGCGGCTAAAAAGCAGAACATCTACGAACGGATTCCATACCGACCCTCCAGACAGAAAGATAACTCCCCGTTGATCGAACCGACGGGCACAGACGACGAAGAGGACGAGGACGACGACGTTTAACGAGGAAGACGAGAACGTGTTTTGCACCATGCAGACCTACAGCAACTCCCTCACGCTTGTCATAGTCACGTCGCTGTTTTTATTCACAGCTCAGGGAAGTTTATCGAATGCCGTCGAACCAATCAAAAAACCCCTAAAGCTCGCCAACTACCGCGCCACTTGCGAAAACCGTACACGCACGCTGGTTACCAGGCTTAACACTAGCCATCACAGCGTAGTCTGGCAACGTTATGATATCTACAGCAGATACATGCGTCGTATGCCGCCACTTTGCATCATTACAGACGCCTATAAAGAAACCACGCGTCAGGGTGGCGCAACTTTCACGTGCACGCGCCAAAATCTCACGCTGTACAATCTTACGGTTAAAGATACGGGAGTCTACCTTCTACAGGATCAGTATACCGGCGATGTCGAAGCTTTCTACCTCATCATCCACCCACGCAGCTTCTGCCGAGCCTTGGAAACGCGTCGATGCTTTTATCCGGGACCAGGCAGAGTCGGTGTGGTCACGGATTCCCAAGAGGCAGACCGAGCAATTATCTCGGATTTAAAACGCCAGTGGTCCGGCCTCTCACTCCATTGCGCCTGGGTTTCGGGACTGATGATCTTTGTTGGCGCACTGGTCATCTGCTTTCTGCGATCGCAACGAATCGGAGAACAGGACGTTGAACATCTGCGGACGGACCTGGATACGGAACCTTTGTTGTTGACGGTGGACGGGAATTTGGAATAAAAGATGCGTAACACCTGTCGAAGATGCGATAACTTTACATACAGGCAAACAGTGTATACAATTATAGTATTTTGTATGTTGCATAAAGTTACATGCAACAGTACTGCTAACAGTACTGCATCCATTACGCTATCCAACACTGCCTCTACCACTTTTGTAACCAACATATATTCAACTCCGAATAACAACACATCAACGACGCCACACACATCTGTCACCTCACAAGCGTCAACCATTGGCAACATCACCAACGTTACCTCCGACTTGAGTACTTTCACAACCGTATATTCTACATTCAATACATCATTTGCCAATATATCTAATACGGCTGTCACTACAGAATTGATTTCAACAAATACCAACACTATCTCATCTTTTACCAACGTAACAGCAAACGCTACATCATCTTATAACACAACAATCACCGTAACTGTCACGTCAGATGAAACTTCGCACAACGTATCCACTAATAATGCACTTATAAGCACACCATGGCCTACAAATTGCAGCGCCACAACATACACCACGTACAACCTTACTAACTCTTCCAACGCTTGTCACACAGAGACAACAATCATACGTTTCAAGGAAACCAATACAACAGGAATAGAAGGGAGTAATGTCACCATAAAGGGTAATTCTACGTGGGACTGTCTTTCAGTCGCCTGGATACGACATTACAATAGATCCACACACGGACATCATCTAGGTTATCGTAAGAACGCACATACCCAATCTTGGTATTGGCTACGCATCCTTACCTCTCACACTGTATGTCATTCTCAACATGAAAGACCTTCACTGTACCATGACTTATGTCGTTCGTGCAACAACACAGAATTACATCTGTACGATCTAAATATCACCAATTCCGGCAGGTACAGCAGACGTTGTTTTAAAGAAAATTACTTCACAGGACATCACGAAGATGAAAATTTCTACCTATTAGTAACACCAAAAAATCATACTGAAGCTATTAATGCTACTTTCGTTTGCCCTAGATACAACACCGATATCGAAAATGAAGATAGAGAGAAAGGAAGTCAACATACTAACAATACACATCACCACAAACGTAATCTCTATCATAGCTCGCAAAGAAGCCGCACCGTATGGACCATCGTGTTGGTTTGTATGGCCTGCATAGTTCTGTTTTTTGCACGACGAGCCTTTAACAAAAAGTATCATATGTTACAAGACACCGTCAGTGAATCAGAATTCATTGTTCGATATCACCCAGAACATGAAGATTGAGCTACGTTTCCGGGCAGACATCTTATGAAGCTGAACAATAAACTAAAACATTCTGTAAGACTCAGCGTTCAAAGGAATATTAATGCCCATTGAGCGAAAACTAATATTGCAATGGACTGGCGATTTACGGTTACGTGGACGATACTAATGTCCGCGTTGTCAGAAAGCTGCAATCAAACCTGTTCTTGTCAATGTCCCTGTAGTACTACCGTTAACTATTCAACTAGTACTGAGACAGCCACATCAACATACAGTACAACAGTTATCAGCAATAAAAGCACTTCAGAATCTATAAATTGCTCTACTGCAACTACACCAGCAAACACCGTTTCTACAAAACCGTCGGAAACAACCACACAGATATCCACAACGACGAACACAAACGTTGAGACTACCACATGTACCAACACCACCACGACCGTTACTTGTGATGGTTTCAATTATACAGTCCATAAAAGATGCGATCGCAGTTACGAGGTAATCAACGTAACAGGATACGTTGGTAGCAACATAACTCTAAAAAAATGCAATCAGACTGAGAAATGGCACAATGTAGACTGGATTCATTATGAGTACCCCACGCATAAAATGTGCGAATTAGGCAACTATCACCAAACCACACCACGGCACGACATATGTTTTGACTGCAACGACACCTCCCTAACTATCTACAACTTAACCACAAAAAACGCTGGAAAATATACCAGGCGTCACCGTGATAACGGTCAAGAAGAAAATTACTACGTAACGGTGTTAATTGGAGACACAACGTTATTCACTCTTGGCACATGCCCTGTAAGATATAAAGAATCTACGAACACTGAAAACACCATTGGAAGTAGCATCATAGAAACCATTGAGAAAGCTAACATTCCCCTGGGAATTCATGCTGTATGGGCAGGCGTAGTGGTATCAGTGGCGCTTATAGCGTTGTACATGGGTAGCCATCGCATTCCCAAAAAGCCGCATTACACCAAACTTCCCAAATATGATCCAGATGAATTTTGGACTAAGGCTTAACATGCTGATCAATAAACTTTTTTTAACCAATAACATGTCTCCGTTTTTTTTTGTTAACAACCTATGATATAAAGCGTTATATTCAGTCGTTACTAAACAAAAAAACATGGGCATGCAATGCAACACTAAATTGTTATTGCCAGTCGCACTAATACCGGTTGCAATCATCCTAATTGGTACTCTAGTGCCGATACTTTTACATGAACAAAAAAAGGCGTTTTACTGGCGACTTTTTCTGCAAAGTCAACATGTAGAAGCACCCATTACAGTAACGCAGGGAGACACAGTCTACCTAGACGCTAGCAATAATCCCTGTAATTATTCCAGCTTTTGGTACCACGGTAATTGCGAACTTTGTGGATGGAACGGATATCTACGCAATGTTACACATTACTACACAAACACATCGTGTTCCCCGCAATTCATCTGCATAAACGAAACTAAAGGTCTGCAGTTATATAATGTAACATTAAACGATTCAGGCGCTTATACTGAACACGTTTACGAATGTGACCTTTCGTGTAACATTACTACTAATAACGAATATGAAATACTCAATTATTTTGATAACTGTAACTACACCATAAATAGCACCAAGCATATTATCACCGTGGTGTCTTCACGTCATTCTAAACAAACAAATTCCCACGTATCCACTCACGCTGGTTGGGCAGTCGCCGTGGTGACGGTAATTATGATCTACGTTCTGATCCACTTTAACGTCCCGGCAACTCTGAGACACAAACTACGAACTAGAAACAACGTAAATCGCATAGCGTGATTATAAAGTATCGACGCTAATTTCTCCAAGATAAAATTTGATTACTCCGTGCAGTTCTCAAAAACTGTAAGGCCCCGCTTTTCCACTCCGTCATGAAGGATCGCAATAGAATACTGCTATGTATCATCTTTATTTGCATTATGTGCCTCATTTGTATTTACTTTAAACGTCGTTGTGTTTTTACTCCGTCTCCAGACAAAGCAGATCTGCGAGTGGAATTTCCCTCGTTACCCCCGTGTATTGGCATACAGTGCGCTGCATGAGAACACGCGTGACACATAGCGTACCCCTGGACGGTACAGTTTATGATAACGTAATTCAGGGAAAGTATACATTCATACCAACATGTTATCACATAACACACAGATTTTCTGCGTGTTTTATAAAAGAGCGTCTCGAAGCAGCTTGAGCCACACTACGGTCCAGATGACGAGCGTAATTAAAAATATGCCGCGCAGTATTCGAAAGCCGTACTGAGCGTGCGAGGCGGGTAGGGTGCCGAACGACGGATATGCGTCGTTGTCATCTTCGACTATAAGGATCGCGACCGAGTCTTCGGCCATGGTAAACGTCACCCTGTGTGGCTGGTATGTAGCGTATCCGGTTTGGAATTGTTCTGCTCCAGCTCGGGGGATAGTGAGGAATTCTCAAGGGATACGGGACCCAATGACTGGATAAGAGAAGGGTTTTTCCCCGTAAGATGATCCTCGTATCACATGAGGTCTGGATATGTATAAATGAAGAGTGAAATAGGCACAGGGAATCAGATGCCAGCCTCGTGATGCAGCCGCTGGTTCTCTCGGCGAAGAAACTGTCGTCTTTGCTGACTTGCAAATACATCCCGCCTTAAGCGATGAGTCTATAAAGCACCGTTGCCCGAGTACGGTAAAAGTGACCCGGATTGTAGAACGTCCTTTTTTTTTGTTTTTGCATCGTTTATCGTCACTACTAGTGCAATATTTTGATTGTAAGGCTGAAAGAGTATCGTTATGATGCTTAGAACGTGGAGATTATTACAGATGGTACTGCTTGCCGCGTACTGTTATTATGTTTTTGCGACTTGTTCAATCAGCACGACGACTGCTCCTGTGGAATGGAAGTCTCCCGACCGTCAGATTCCCAAGAATATTACCTGCGCTAATTACTCAGGGACCGTCAACGGCAACGTTACATTTCGAGGTCTTCAGAACAAAACGGAAGACTTTTTGTACTGGTTGTTAGGATGGGGTCATAAGTCCATTTGTTCGTTCTTCCCGAAACTCCAGGGTAACTATGACGAACAACATTACAGATATGAAGTAGCGAACCTGACGTATAACTGCACCTATAACCGCTTGACGTTGCTGAATCTGACGACGGAAAACAGCGGAAAGTACTATTTCAAAAGGGAAGATGCGAATTTCACCTTCTATTACTCTTGTTACAACTTGACCGTGTCCTAAAGATCGCACGTGAAGTTTCACAGAGCCGCGTGGCTGTAGCTATTGTGTTTACGTTGCTTTTGAAATGTTAAGCGTCCCTACGGCGCTAACATGTTTCTAGGCTACTCTGACTGTGTAGATCCCGGCCTTGCTGTGTATCGTGTATCTAGATCACGCTTAAAGCTCATGTTGTCTTTTGTGTGGTTGGTCGGTTTGCGTTTCTATGATTGTGCCGCGTTCGAGTCCTGCTGTTACGACATCACCGAGGCGGAGAGTAACAAGGCTATATCAAGGGACGAAGCAGCATTCACCTCCAGCGTGAGCACCCGTACACCGTCCCTGGCGATCGCGCCTCCTCCTGACCGATCGATGCTGTTGTCGCGAGAGGAAGAACTCGTTCCGTGGAGTCGTCTCATCATCACTAAGCAGTTCTACGGAGGCCTGATTTTCCACACCACCTGGGTCACCGGCTTCGTCCTGCTAGGACTCTTGACGCTTTTCGCCAGCCTGTTTCGCGTACCGCAATCCATCTGTCGTTTCTGCATAGACCGTCTCCGGGACATCGCCCGTCCTCTGAAATACCGCTATCAACGTCTTGTCGCTACCGTGTAGCTAGTTAGCCAGCTGTGTGTAGTGTTTTGCTTTTGCATATTTGTTTTCAGTCAGAGAGTCTGAAACGGGGTGGGAGGGACTTTTGCGGGTAGTGCATGCTAAGATGAACGGGTGGGCTGGGGTGTGCTTGATAACTCACTGTTTGAATACGCGCTCACGCACATATGTAGCACTCAACATGTTAGCTTTTGCCCGCACGCCCCGGGGCGTGCCGAGCTGCCTTTTTAATAAAGTCTGGGTTTCCAGATACGCGCTGGTTCTGATTTTGATGGTTTGTGCCTCTGAAAGCTCTACGAGCTGGGCCGTGACATCCAATGGACTGCCTAACTGTAGCACGGTAACTAGAACAGCGGGTCAAGACGCTGAATTGCACGGTCCGGCACCGTTAAGCTGTAATGTGACCCAGTGGGGACGTTACGAGAATGGAAGCACACCCGTGTTATGGTGCACTTTACGGGGATCAAGCATGCGAGTCTCATTAGGACACCGTGTAGCGTTTGGCTGTTCTTGGAAAACATTTTTTATTTATAACGTTTCTGAAAGTAGCGGTGGCACTTACTATCAAAAAGGTTACAACTGCACCGACAAACATATAACACTATCTTGTTTCAACTTAACGGTGGTTCCTCGAGCGGTTCAAAGCACAACCACCGTAATGACACCCACGCTGGTTACAAACTCCACATTCAGTGTGTCACTTGTTCCGTTGAGACTGACGACAAATTCCAGCGCGTTTGGACACGCTATTTATCAACGACAACAGCGTGTTGAAAACGGGACGTTATCCAAGAACATAACTAACTTGGCATTCACCTATGGCAGCTGGGGCGTTGCGATGCTGCTGTTTGCCGCCGTGATGGTGCTCGTTGATTTGGGTTTGCCTCAATCGGCTTGGCGACGCTGGCGAAGCCACGTGGACGATGAAGAACGTGGTTTGTTAATGTAGGAAATAAAAGGCAGTTTGAGCATGACTGTTTCCAAACCGTAACGTGGTAAATAAATCATGGCTTCCGACGTGGGTTCTCATCCTCTGACGGTTACACGATTTCGCTGCAGAGTGCATTATGTGTACAATAAACTGTTGATTTTAACTTTGTTTGCCCCCGTGATTCTGGAATCCGTCATCTACGTGTCCGGGCCACAGGGAGGGAACGTTACCCTGGTATCCAACTTCACTTCAAACATCAGCGCACGGTGGTTCCGCTGGGACGGCAACGATAGCCATCTCATTTGCTTTTACAAACGTGGAGAGGGTCTTTCTACGCCCTATGTGGGTTTAAGCCTAAGTTGTGCGGCTAACCAAATCACCATCTTCAACCTCACGTTGAACGACTCCGGTCGTTACGGAGCAGAAGGTTTTACGAGAAGCGGCGAAAATGAAACGTTCCTGTGGTATAATTTGACCGTGAAACCCAAACCTTTGGAAACTACTCCAGCTAGTAACGTAACAACCATCGTCACGACGACATCGACGATGATCGACGCGAAAAGTAACGTTACAGGGAACGCCAGTTTAGCACCACAATTACGTGCCGTCGCTGGATTCTCCAATCAGACGCCTTTGGAAAACAACACGCACCTGGCCTTGGTAGGTGTTGTTGTGTTTTTAGTTCTGATAGTTGTTTGCATTATGGGGTGGTGGAAATTGTTGTGTGGTAAACCAGAGTTATAGTAATGTGCTTTTTATCAGGGAGAAGGTTTTGTGCCAACAATGACTAGCCCGGGACTATCTGCGTCAGAAAATTATGACGGAAATTATGAATTCACGGAAACCGCCAATACAACGCGTACAAATAGAAGTGACTGGACAACGTTAGAAACCAGTGCATTGCTATTGAAAAACACGGAGACTGCAGTGAACCTCAGCAACGCGACTACGGTCATCCCACAACCTGTAGAATACCCGGCTGGGGAAGTACAATATCAAAGAACGGCAACGCATTATTCTTGGATGCTAATCATTGTCATCATTCTCATCATTTTTATTATCATCTGTCTACGAGCACCTCGAAAAATCTACCATCACTGGAAAGACAGTAAACAGTACGGACAAGTGTTTATGACAGACACGGAACTGTGACAGTGATGTCTAAGCGTTTGCAGGTATTTCCATGGATAACAATTTTATTTTACACATCAAAATCCCAGTATTGGAACTATATGGCAATACCATGTACCCCTACAGTTGGATACGGCAGTCATAATATTAGCTTGCATCCGCTTAATAACTCATTATTTCAAGACGATGTTTTTGAATGGTACATAGACAAACCAATGGTTACAAGTTATGTCTTTATCAAAGTAATGAACGCACAAAATCCAATCTAGACTCTCCAAATATTGTGTGGCAATGCACAGATAATCGTACACTAATTCTCATGAACTTAACCACAACATACAGTAGAAACTATTATTTTCAATCCTTTAAATATCTCGGACGAGGAGTACCAAAACCGAATAACTTGTGTTATAACGTTAGTGTACACTTTACCCACCAAACACATTGCCATACAACTACATCATCCCTGTATCCACCTACATCTGTACACGATTCATTAGAAATATCACAGTCATTCACCTCAACCAACTTCACACATACCGCGGTCCACTACGCCACCGGTAACGTTGAAGCACAACACGACACTACCACTCCACATACAATGTGGATCATACCCCTAGTTATCGTTATAACAATCATCGTTTTAACTTGTTTCAAATTCCCCCAGAAAGCTTGGAATAAATTCACACAATACAGATACAGCGGTATGCTCGCCGCCGCTTAAAGAATCAACGCCAAGGAAACCAAAACGTAAAAAGAATAGATATGTACGTTTATTTTTCAGCTCACTGTTTGAATACCGTAAACATAATGACGTACATATACGTGGTTATACAACAGGTGTTTGTGTTATGCGGCGACTGATTAACCATATCGTGAACCATGATCTTTTCCGATGGTCCGTCGTGACCGCAATGATATTTTACAGATATTCCGAAACCTGTATGGAGGTCACTGTCAGAGTAGGTGATCCAGTTACCCTCGGTAGTGGACATGGTTATCATCCAGGTAGGGATAACAGGGTAATGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGSEQ ID NO:  12 DNA Genus/species-Phikmvlikevirus LKA1Descriptive title-LKA1 gp49 sequenceATGGCGCAAACACCCAGTACATGGGCCGACTACGTAGGCGACGGCGTAGAGGATACGTTCCAAGTCACATTCCCGTACCAGAAGCAGCAAGAGGTGTTTGTGACTGTGGGCGGCGATCCGGCAGCTTTCACATTCATCTCGGCAGGTTGGATTCAACTGGCAGCGGTCCCGGTAAATGGGGCCGCAATCCGTGTACGGCGCAGCACTGAGGCATTCGAGCCTCGGCACGAGTTCGCCAACGGCGTGCCATTACTGCCGCGATTCATAGACGAGAATAATACCCAGTTCTTGTACACTGTACAAGAGGCAGTGAATGAGACACATGGCATTGCTTCCGAAGCGCTGAGTGTCGCAGAGGAGGCCAGAGGCATTGCGCAGGCGGCATCGGATAAAGTGGATGCTGCCACCATTGACTCCGCACACCAGTTGCGTCTAGACCTCGCCGACCCGGCGAAGGGGCCTGGGCTGCTAGGCTACGACCGAGACGTAAGTTATCCGGTCGGGTCGGTCGGTCAAAGCCTACAGTTTCTGGAAATGGGTCGGGTCACACCAGCGCAATTTGGCGCCGTTGGTGATGGCGCCAGCCACCCCCTCTCTGAGCGATACGCAACTCTAGCGGAAGCTCAGACTGTCTATCCGCATGCAGTCGCACTCTCCGACGAAATAGACTGGGCCGCATTGCAAGCTGCCGTGGATTCAGGGGCACCTGTACACATACCGTCTGGGGACTATCAGATAAATAGGGGGATTAGCAGTACGGGCTCTCTACAGATTGCGGGTGATGGCGCTACATCTATTATACGCCCGACTGCTGCGTTCACTGGTACATCGGTCCTCAGTTGTGTGGGGAGCTTAGTTGCCTTGCCGAATATATCCTCCGTGTCGGCTGGGTCCCTAACCATTGACTTTGCCAGCACCCCTAATCTTGTAGCGGGGGATGTATTCATCATCTACAACCCGACTGATAGCAGCTTCTCGGGATTTCGGACGAGCTATCGCGCAGGAGAGTTCTGTGAGGTCAGGGCGGTTTCTGGGAACACCGTGACAATCCGTTCCGCACTCTATGCCGCATACGACGGGGCTACTGTTGCTATTTACAAAGTAGTCTCTGGTGTAGTTGATATAGCTAGCATCCAAATCGTTGGCGGGACAGTCCCAATGAATGGACTGTTAGTGGAGGCTGTCGTTTCACCGCGCGTCGATGACGTGACGGTCACCCTTGCAAACAACGCCGGTGTGTATTTTGCCCGCTGCTATGACGCTAAGATCACAAACAGTAATATATCGAACATCGGCGACGGTGGCGATGACTATGGAATCATCTTTGGGAACTGTCACGACGGTGGGGCAGACAACTGTAAAGTCTACGCTAGGCGACATGCCATCGCCACGGGCGGCGATGCAGAAGTAGGCTGCGTTCCGGTCCGTAATGTGCGTATGCGTAACTGCACACTTAGGAATGATATTACCTCTGGTACACACTGCGCAGACTTCCACGGTAACGCCGAGGATTGCAGCTACGAAAACTGCACAATCTACGGTGGTGCAACTTGGCAGGGGAAGGATATCAGCTACAGACACTGTACAATCACTAACGCGTCGGGTGGTTGGATTGTTATATCCGCTGAGATTCTTGGTGGTACATTCCTTCTCGACCAATGCACATTGTACACAACCGGCGATCCGCAGCCTGGTAACCGTGGGGTTATAGATGTAGGTGGGAACTCCGCAGTCCTCACTACAAATACAACGCAACCCTGTAACTTCCTTATACAAGGCGGCAGTCTGCGAGCGCCCAGCTTAAGTACGTCTAGTTACCTACTGCGCGCACGTCTTGAGGGTAGTACAGTTCCAGTAAACATACAGTACAGCGGACAGGCTATTGATGTAGGCTCTCTGGGCAAGGTACTACAACTCGATATTACCTCGGGCAGTACCTCTCCTGAGTATTTGATCGTGGAGAATTTAGCGGGGTTGCCATCTGGCATCACGCTGGCGTCTGCTGCTGGTGGTTTCGCAAGTGCCCCGATGCGTATGCCTGTGCTGGGTGGTAGGGTTCAAGTAACTACGGCAACCAACGCGAGTAGCGTTACTGCTCCAGTAACGTTCAGGTACATTTATCCTAAGGCCCCAACCGTCCAGGTCACAAAGACGGACAGGAGCTACGCCGGTAACAGGGTCGGCGTTGCTATCGCCAATCCGACCTCTGCGTCTGGGGCGACGTTGGGTCTGTTCACGGACGACGGGACAAACTTTAGCTCAGCCGTTACTAACCAGTTGAACTGGCAGGCAGGTATTTATGAGGTGTAASEQ ID NO: 13 DNA Genus/species-Phikmvlikevirus NTUH-K2044-K1-1Descriptive title-NTUH-K2044-K1-1 gp34ATGGCCCTGATCCGGCTCGTGGCGCCCGAGCGCGTGTTCAGCGACCTGGCCAGCATGGTCGCCTATCCGAACTTCCAGGTGCAGGACAAGATCACCCTGCTGGGCTCGGCCGGCGGCGACTTCACCTTCACCACCACCGCGTCGGTGGTGGACAACGGCACCGTGTTCGCCGTGCCCGGCGGCTATCTCCTGCGGAAGTTCGTCGGCCCGGCGTATAGCTCGTGGTTCAGCAACTGGACCGGGATCGTCACGTTCATGAGCGCGCCGAACCGGCACCTGGTGGTGGACACCGTGCTGCAGGCCACGAGCGTGCTGAACATCAAGAGCAACAGCACGCTGGAATTCACGGACACGGGCCGCATCCTGCCCGACGCCGCCGTGGCCCGCCAGGTGCTGAACATCACCGGCTCCGCGCCCTCGGTGTTCGTGCCCCTCGCCGCCGACGCCGCCGCGGGGTCGAAGGTGATCACCGTGGCCGCCGGCGCGCTGTCCGCGGTGAAAGGCACCTACCTCTATCTGCGCTCCAACAAGCTGTGCGACGGCGGGCCGAACACCTATGGCGTCAAGATCAGCCAAATCCGTAAGGTGGTCGGCGTGAGCACCAGCGGGGGCGTGACGTCCATCCGCCTCGACAAAGCCCTGCACTATAACTACTACCTCTCGGATGCCGCCGAAGTGGGCATCCCGACCATGGTGGAGAACGTCACCCTGGTGAGCCCGTACATCAACGAGTTCGGCTACGACGACCTGAACCGCTTCTTCACCAGCGGCATCTCCGCGAACTTCGCGGCCGACCTGCACATCCAGGACGGCGTCATCATCGGCAACAAGCGTCCGGGCGCCTCCGACATCGAGGGCCGCAGCGCCATCAAGTTCAACAACTGCGTGGATAGCACCGTGAAGGGCACCTGCTTCTATAATATCGGCTGGTACGGCGTGGAGGTCCTCGGCTGCTCGGAGGACACCGAGGTGCACGACATCCACGCCATGGACGTGCGCCATGCCATCTCCCTGAACTGGCAAAGCACCGCCGACGGCGATAAGTGGGGCGAACCGATCGAGTTCCTGGGCGTGAACTGTGAGGCGTACAGCACCACCCAGGCCGGCTTCGACACCCACGACATCGGGAAGCGTGTCAAATTCGTCCGCTGCGTGTCCTACGACAGCGCGGATGACGGCTTCCAGGCCCGCACCAACGGCGTGGAGTACCTCAACTGCCGCGCCTACCGCGCCGCCATGGACGGCTTCGCCTCGAACACGGGCGTCGCCTTCCCGATCTACCGCGAATGCCTGGCCTACGACAACGTGCGCAGCGGGTTCAACTGCAGCTACGGCGGCGGGTATGTGTACGACTGCGAGGCGCACGGCAGCCAGAACGGCGTCCGCATCAACGGCGGCCGGGTCAAAGGCGGGCGCTACACCCGCAACTCGTCGAGCCACATCTTCGTGACGAAAGATGTGGCGGAAACCGCCCAAACCAGCCTCGAGATCGACGGCGTCTCCATGCGGTACGACGGCACCGGCCGCGCCGTGTACTTCCACGGCACCGTGGGCATCGATCCGACGCTCGTGAGCATGTCCAACAACGACATGACCGGCCACGGCCTGTTCTGGGCCCTGCTGTCCGGCTATACCGTGCAGCCGACCCCGCCGCGCATGTCGCGCAACCTGCTCGACGATACCGGCATCCGCGGCGTCGCGACCCTGGTCGCGGGCGAAGCGACCGTCAATGCCCGCGTCCGCGGGAACTTCGGCAGCGTGGCCAACAGCTTCAAGTGGGTGTCGGAGGTGAAGCTGACGCGCCTCACGTTCCCGTCGTCGGCCGGCGCCCTCACGGTCACCAGCGTCGCCCAAAACCAGGACGTGCCGACCCCCAACCCGGACCTGAACAGCTTCGTCATCCGCAGCAGCAACGCCGCCGACGTGTCCCAAGTCGCCTGGGAGGTCTACCTGTGA SEQ ID NO: 14 DNA Genus/species-T7-like Pp15Descriptive title-Pp15 gp44 sequence addedATGGCACGAACTATCGTCCAGAACGCCCTAACAGGCGGACAACAGGACTTCGAGGTACCTTTCGACTACATCTTGCAGCGCTTCGTTAAGCTTACCCTGATCGGTGACGGTAACCGACAAGAGCTGGTCCTCGGTACCGACTTCCGGTTCATCGGTCCTCGCACCGTTCGCACTAACGTCTTCTGGGGACCAGCGCAGGGGTATACCTCCATCGAGATCCGACGAGTTACCAGCGCTTCTGATCGTCGCGTAGAGTTCTCGGACGGGTCCATCCTGACCGCAGGTGATCTGAACATCGCCCAGCTTCAGGCCATCCACATTGCCGAAGAAGCGCGAGACTCTGCCACTGAGAACCTGAGCCCAGATGCTGATGGCAACTACGATGCACGTGGTGCGCGCATTTACAACCTCGGTGACGCTGTTCAGCCGAAGGATGCGGTCAACCGGTACACTCTTGACCTCGCTATCGCAGCCGCTCTGGCCATGAATACCGGCAACCCGAACAACGCCCAGAACATCTCGTACACCCCTAACGGGCCTGGTCAGTCGATCCGAAGTGTTGAAGGCCGTCTGCGGGATGCTGTGTTCGTCTCGGACTACATGACCACTCCACGTGATGGAGTTACCAGTAACCAGCAGGACCTCGAAAAGGCACTCGCTGCGGCGAACGCTAAAGGTGCCGACCTATTCTGGCCTGACGACATCCCGTTCTTCTCCACGTCCCCGCTGGCACTGATCCACGCGGTCTACCATGTTGGACGTGGTGTCATCAACGCGAACGGTACGCTGTTCTACGTGAACCCGAAGAACGGCCAACACAACAGGCTACACGTGTCTCCCGGGGGCACCGGGGATGGTCTGGCAGCTGGCCGCCCACTGGGGACCATCTGGAGTGCACTCGCGGCCCTTAACATGCGAGCCCCACTGACCACGCGCTGGTCCTTGGAGATGACCGCTGGCGCCTATAATGAAGCCGTTACACTTCCGAACTACCTGACCAGCTGTAACGACTACTTGGCGTTTAACTGGCCGAACACCGGTCAGGAACGTATGGAGCCCACTGCGTACCCATCAGCTCTCGACGGCACAGGCCAGACCGGCCTCACAGGTTTCCACACTGGCATCGGCAACCGCATTACCATCAACAACGTGTGCATGTCCAACTGGTACGACACTGCGCTGACTCCTACCCAACAGGTGCGAAGAGCGTTCGTTGTAGGTGCGTATTCGACTGCCTACGTGGTCAACTGCGCGTTCATTTACAACGGCATCGCGAGCGTGTCTGTGCTGCCCGGTGGCACTGCTATCGTAACCGGTGGCATCGTCGATGGTGGGCGGTTCGGCCTCGACAACACTGGCGGTCGCCTGTCCCTGACGGCAACCAAGAGCAATTATACGCAGGTCCGGAACTGCCTCGAATATGGACTGTACTCGAAGCATGACGCATCGACCGTAATGGACAACACCGAGTTCCGCAACTGCGGTAATCACCCTGCGGCTGTTGCGTATGGTGCTGCAATCTTCGCGTACAAGTTCAACTGTTCTGTTGACACTCGTGGGGTCAAGTTCTACGGCAACAACATCGCCCAGCACTGCCGTGGCGGTATCACCTCGGACAATCCGGGCGATCCGGACATCTACGGTACCGGCGCAGATGCTAATAAGCGTCTATTCCTGTGCACCGGTGGTGGCTCTGACGACATCCAGTTCTACGAAGCTCGGCGCGTCATGGACATCACGAAGCGCACTGGTGGCGGCTCAACTACTGCCAGCGTATCGTCGCTGCTACTGGCTGCCGTTGCGTCTGTCCGTAAGGGCTACTTTGCGCACAACGATCAGGTGATCCGGATGACCCTGATGTTCCGCGCTACAGGCTCGGCTGGCATCTTCACGCCGACCTTGCGCACACCTCTGGGGACTATCCCTCTGGGTAGCTTCAGGGTCGCATCGGGACAGTACGGCGAGATCAAGTTGACCATTCGACCTACTCTGACATCTGATGGTCTCATAGTCGGGTTCTCCTGCATCAACGCCGTGCAGAATCTTGGGTCCTCTGTTGGTCAAATCATCGTCAGCGGCACCGTAGACCTCCGCACCGTCGACCAGCTGGTCGAGATGTGGGGCTATTCGGAAGCTGGTGGCACCGCTTCGTACATTCAAGGCCTGATCGAGCTGGTCGGGTGASEQ ID NO: 15 DNA Genus/species-Aggregatibacter actinomycetemcomitansDescriptive title-dspB sequence addedATGAACTGTTGCGTCAAGGGCAATTCCATCTACCCCCAGAAGACCTCCACCAAGCAGACCGGCCTGATGCTCGATATCGCCCGGCATTTCTACAGCCCCGAGGTGATCAAGAGCTTCATCGATACGATCAGCCTGAGCGGCGGCAACTTCCTCCACCTGCACTTCTCGGACCATGAAAACTATGCCATCGAGTCGCACCTGCTCAACCAGCGGGCGGAGAACGCCGTCCAGGGGAAGGATGGCATCTACATCAATCCGTACACCGGGAAACCGTTCCTGAGCTACCGCCAGCTGGACGACATCAAGGCCTACGCCAAGGCCAAGGGCATCGAACTGATCCCGGAGCTGGACAGCCCGAACCATATGACGGCCATCTTCAAACTGGTCCAGAAGGACCGCGGCGTCAAGTACCTGCAGGGGCTGAAATCCCGCCAGGTGGACGACGAGATCGACATCACCAACGCCGATAGCATCACCTTCATGCAGAGCCTGATGAGCGAGGTCATCGATATCTTCGGCGACACGAGCCAGCACTTCCACATCGGCGGCGACGAATTCGGCTACTCCGTCGAGAGCAACCACGAGTTCATCACCTACGCCAACAAGCTGTCGTACTTCCTGGAGAAGAAGGGGCTCAAGACCCGCATGTGGAACGACGGCCTCATCAAGAACACCTTCGAGCAGATCAATCCCAACATCGAAATCACGTACTGGTCGTACGACGGCGACACCCAGGATAAGAACGAAGCGGCCGAGCGCCGCGACATGCGCGTGAGCCTGCCGGAGCTGCTGGCGAAGGGCTTCACCGTGCTGAACTACAACAGCTACTACCTCTACATCGTGCCGAAGGCGAGCCCGACGTTCTCGCAGGACGCCGCCTTCGCCGCCAAAGACGTGATCAAGAACTGGGATCTGGGCGTCTGGGATGGCCGGAACACCAAGAACCGCGTGCAGAACACCCATGAGATCGCCGGGGCGGCGCTGTCGATCTGGGGCGAGGATGCGAAGGCGCTCAAGGACGAGACGATCCAGAAGAACACCAAAAGCCTGCTCGAGGCCGTCATCCACAAGACCAACGGCGACGAGTGA SEQ ID NO: 16 DNAGenus/species-Staphylococcus aureusDescriptive title-SaPSMa3 sequence addedATGGAGTTCGTGGCGAAGCTCTTCAAGTTCTTCAAGGACCTGCTCGGGAAGTTCCTGGGGAATAACTGASEQ ID NO: 17 DNA Genus/species-Staphylococcus aureusDescriptive title-SaPAMb2 sequence addedATGACCGGCCTGGCCGAGGCGATCGCGAATACCGTCCAGGCGGCCCAGCAGCACGACAGCGTCAAGCTGGGCACCTCGATCGTGGACATCGTCGCCAACGGCGTGGGCCTGCTGGGCAAACTCTTCGGCTTCTGASEQ ID NO: 18 DNA Genus/species-Staphylococcus epidermidisDescriptive title-SePSMa sequence addedATGGCGGACGTCATCGCCAAGATCGTCGAGATCGTGAAGGGCCTGATCGACCAGTTCACCCAGAAGTGASEQ ID NO: 19 DNA Genus/species-Levivirus MS2Descriptive title-MS2 L sequence addedATGGAGACCCGGTTCCCGCAGCAGTCCCAGCAAACCCCGGCCAGCACCAACCGCCGCCGCCCCTTCAAGCACGAGGACTACCCGTGCCGCCGGCAGCAGCGCAGCTCCACCCTGTACGTGCTGATCTTCCTGGCGATCTTCCTGAGCAAGTTCACCAACCAGCTGCTGCTGTCCCTGCTGGAGGCGGTCATCCGGACCGTCACCACCCTGCAGCAGCTGCTGACCTGA SEQ ID NO: 20 DNA Genus/species-Levivirus PRR1Descriptive title-PRR1 L sequence addedATGTGCAAGGTGTCTACTAAGGTAGACTCTAAACTGACTGAGTCAGTTGGACAACTCACCATAAGGAGCTACCTATGGCTACGGAATATCCTAGCATTAGCAGGACTTCTTTTCGTAATCCTTCTTGCGACCAATCATTTATCCATCGCTATCTACAGTCCGTAA SEQ ID NO: 21 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 gp32 promoter (P32)CGACCCTGCCCTACTCCGGCCTTAAACCCACATCCAAAAGAGAGAGAATCGC SEQ ID NO: 22 DNAGenus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 gp32 terminator (T32)TGCCACGAAACCCCGCACTTCGGTGTGGGGTTTCTTCAAAGCCTAACGACCCGCGCAGATTCCCTGCGTGGGTTTTTGCGCTTTAGGAGAAACCCT SEQ ID NO: 23 DNAGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp7 regionTACAAGGTGGTGGCACCCAGCTCGGCGGAAGGTATCATTGTGCTGGCGACCAAGCAGACGCCGGCGCTAGCCCAAGCAGCCGTCGTACTGCACAGCATGAACCCTGCGCAGTATCCCGCAGGTTCGGCTATCCTCAACACGGCCTGGAAGTGCCGCCGCCTGGGAGTGGGCGAGTACGTCAAGCTCGTCCAAGGGGAGGAGGAC SEQ ID NO: 24 DNAGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp18 regionGAATGCCAACCGAAGAAGAACGCATGATCCGCTGTTTACTGGCGGATATCCACGAGCCACTGGACCTGCTGTTCCCCGGCCTCCGTACCAAGGCCCATATGGACCCGCAAGCAGAGGAACTGTCGATTCGAATTGACTACGACCATGCGAAGCTGGGCCGTATGGGATTCTGCCACGCGGTATCCCTATATCAACTGTCCATATATGGCCGCGAGGGGATGGTCCGCTACCTGATGCAGGAGATTCCCCGCCGCGTGCTGGAAGGTCTGCTGGTCAAGGCGCAGCAGTACAGCCAAAGCAACTGGTACAGCAAATGACGAC SEQ ID NO: 25 DNAGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 gp49 and gp48-gp49 intergenic regionGGGGACACCATGAGCAAAGCCAAACTACGAGTCATCGCCGACACCCCGGAGCTGGAGTCAGTGCTAAAAGCATTGCTGACCGCCACCTACGCTATCGAGGACCTGCTCAACGAGGCCGTGGCTAGCAAGGTGCTAAACTCCCGCCTGGGCTGGTCCGCAGTCGGCGAGTATGTCGAACTGTTCAACCGCACGCAATCCCGCGTGGCCGGGTTGATTCCCGAGTAG SEQ ID NO: 26 DNA Genus/species-Phikmvlikevirus LKD16Descriptive title-Wild type LKD16 gp18 geneGTGCGAGTACCAACTGAACACGAGCGCACCCTGCGCTGCCTGCTCCAAGACATCCACGGGCCGCTGAATCTGCTGTTCCCAGGTATCCGGGTGAAGGTGGAGGAGGCGTGCCTCGGATACTTGGGCTACAGGGAGCGGGGCTATTGGGAGCTGCGCCTCCAGGTGGACTACGACCACCCGAAGCTTGGGCACCTCCGCTACAGTCAGGCCGTGCCGGAGTACGTGCTGATCAACGACCGCGACAGCATCATCAAGTACCTGATGGAAGCAGTCCCTCGGCAGGTACTAGAGGGCATGCTCAATAAGGCCCAGGAATTCGTAACCAAGAACTGGTATTCCCTATGA SEQ ID NO: 27 DNASynthetic (artificial/unknown)Descriptive title-Gene encoding NLS-FLAG-CAS9-HisATGCCCAAGAAAAAGCGGAAGGTCGGCGACTACAAGGATGACGATGACAAGTTGGAGCCTGGAGAGAAGCCCTACAAATGCCCTGAGTGCGGAAAGAGCTTCAGCCAATCTGGAGCCTTGACCCGGCATCAACGAACGCATACACGAGACAAGAAGTACTCCATCGGGCTGGACATCGGGACGAACTCCGTGGGATGGGCCGTGATCACAGACGAATACAAGGTGCCTTCCAAGAAGTTCAAGGTGCTGGGGAACACGGACAGACACTCCATCAAGAAGAACCTCATCGGGGCCTTGCTCTTCGACTCCGGAGAAACCGCCGAAGCAACGCGATTGAAAAGAACCGCCAGAAGACGATACACACGACGGAAGAACCGCATCTGCTACCTCCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACTCGTTCTTTCATCGCCTGGAGGAGAGCTTCCTGGTGGAGGAAGACAAGAAACATGAGCGCCACCCGATCTTCGGGAACATCGTGGACGAAGTGGCCTACCACGAGAAATACCCCACGATCTACCACTTGCGCAAGAAACTCGTGGACTCCACGGACAAAGCGGACTTGCGGTTGATCTACTTGGCCTTGGCCCACATGATCAAATTTCGGGGCCACTTCCTGATCGAGGGCGACTTGAATCCCGACAATTCCGACGTGGACAAGCTCTTCATCCAGCTGGTGCAGACCTACAACCAGCTCTTCGAGGAGAACCCCATCAATGCCTCCGGAGTGGACGCCAAAGCCATCTTGTCCGCCCGATTGTCCAAATCCAGACGCTTGGAGAACTTGATCGCACAACTTCCTGGCGAGAAGAAGAACGGCCTCTTCGGCAACTTGATCGCGCTGTCGCTGGGATTGACGCCTAACTTCAAGTCCAACTTCGACTTGGCCGAGGACGCCAAGTTGCAACTGTCCAAGGACACCTACGACGACGACCTCGACAACCTGCTGGCCCAAATTGGCGACCAATACGCGGACTTGTTTTTGGCGGCCAAGAACTTGAGCGACGCCATCTTGTTGAGCGACATCTTGCGCGTGAATACGGAGATCACCAAAGCCCCTTTGTCCGCCTCTATGATCAAGCGGTACGACGAGCACCACCAAGACTTGACCCTGTTGAAAGCCCTCGTGCGGCAACAATTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGGTACGCCGGCTACATCGACGGAGGAGCCTCCCAAGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGTTGCTCGTGAAGCTGAACCGCGAAGACTTGTTGCGAAAACAGCGGACGTTCGACAATGGCAGCATCCCCCACCAAATCCATTTGGGAGAGTTGCACGCCATCTTGCGACGGCAAGAGGACTTCTACCCGTTCCTGAAGGACAACCGCGAGAAAATCGAGAAGATCCTGACGTTCAGAATCCCCTACTACGTGGGACCCTTGGCCCGAGGCAATTCCCGGTTTGCATGGATGACGCGCAAAAGCGAAGAGACGATCACCCCCTGGAACTTCGAAGAAGTGGTCGACAAAGGAGCATCCGCACAGAGCTTCATCGAGCGAATGACGAACTTCGACAAGAACCTGCCCAACGAGAAGGTGTTGCCCAAGCATTCGCTGCTGTACGAGTACTTCACGGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGCAAACCCGCGTTCCTGTCGGGAGAGCAAAAGAAGGCCATTGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAACAGCTGAAAGAGGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGATTCAATGCCTCCTTGGGAACCTACCATGACCTCCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGAGAGATGATCGAGGAACGGTTGAAAACGTACGCCCACTTGTTCGACGACAAGGTGATGAAGCAGCTGAAACGCCGCCGCTACACCGGATGGGGACGATTGAGCCGCAAACTGATTAATGGAATTCGCGACAAGCAATCCGGAAAGACCATCCTGGACTTCCTGAAGTCCGACGGGTTCGCCAACCGCAACTTCATGCAGCTCATCCACGACGACTCCTTGACCTTCAAGGAGGACATCCAGAAGGCCCAAGTGTCCGGACAAGGAGACTCCTTGCACGAGCACATCGCCAATTTGGCCGGATCCCCCGCAATCAAAAAAGGCATCTTGCAAACCGTGAAAGTGGTCGACGAACTGGTGAAGGTGATGGGACGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCCGCGAGAACCAAACCACCCAAAAAGGACAGAAGAACTCCCGAGAGCGCATGAAGCGGATCGAAGAGGGCATCAAGGAGTTGGGCTCCCAGATCCTGAAGGAGCATCCCGTGGAGAATACCCAATTGCAAAACGAGAAGCTCTACCTCTACTACCTCCAGAACGGGCGGGACATGTACGTCGACCAAGAGCTGGACATCAACCGCCTCTCCGACTACGATGTGGATCATATTGTGCCCCAGAGCTTCCTCAAGGACGACAGCATCGACAACAAGGTCCTGACGCGCAGCGACAAGAACCGGGGCAAGTCTGACAATGTGCCTTCCGAAGAAGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTCAACGCCAAGCTCATCACCCAACGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGAGGATTGTCCGAGTTGGACAAAGCCGGCTTCATTAAACGCCAACTCGTGGAGACCCGCCAGATCACGAAGCACGTGGCCCAAATCTTGGACTCCCGGATGAACACGAAATACGACGAGAATGACAAGCTGATCCGCGAGGTGAAGGTGATCACGCTGAAGTCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCATCACGCCCATGACGCCTACCTGAACGCCGTGGTCGGAACCGCCCTGATCAAGAAATACCCCAAGCTGGAGTCCGAATTCGTGTACGGAGATTACAAGGTCTACGACGTGCGGAAGATGATCGCGAAGTCCGAGCAGGAGATCGGCAAAGCCACCGCCAAGTACTTCTTTTACTCCAACATCATGAACTTCTTCAAGACCGAGATCACGCTCGCCAACGGCGAGATCCGCAAGCGCCCCCTGATCGAGACCAACGGCGAGACGGGAGAGATTGTGTGGGACAAAGGAAGAGATTTTGCCACAGTGCGCAAGGTGCTGTCCATGCCTCAGGTGAACATCGTGAAGAAGACCGAGGTGCAAACAGGAGGGTTTTCCAAAGAGTCCATTTTGCCTAAGAGGAATTCCGACAAGCTCATCGCCCGCAAGAAGGACTGGGACCCCAAGAAGTACGGGGGCTTCGACTCCCCCACGGTGGCCTACTCCGTGTTGGTGGTGGCCAAAGTGGAGAAAGGGAAGAGCAAGAAGCTGAAATCCGTGAAGGAGTTGCTCGGAATCACGATCATGGAACGATCGTCGTTCGAGAAAAACCCCATCGACTTCCTCGAAGCCAAAGGGTACAAAGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAGCTGGAGAACGGCCGCAAGCGGATGCTGGCCTCCGCCGGGGAACTGCAGAAAGGGAACGAATTGGCCTTGCCCTCCAAATACGTGAACTTCCTCTACTTGGCCTCCCATTACGAAAAGCTCAAAGGATCCCCTGAGGACAATGAGCAGAAGCAACTCTTCGTGGAACAACACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGCGCGTGATCCTCGCCGACGCCAACCTGGACAAGGTGCTCTCCGCCTACAACAAGCACCGCGACAAGCCTATCCGCGAGCAAGCCGAGAATATCATTCACCTGTTTACCCTGACGAATTTGGGAGCCCCTGCCGCCTTTAAATACTTTGACACCACCATCGACCGCAAAAGATACACCTCCACCAAGGAAGTCTTGGACGCCACCCTCATCCACCAGTCCATCACGGGCCTCTACGAGACGCGCATCGACCTCTCCCAATTGGGCGGCGACCATCATCACCACCACCACTAA SEQ ID NO: 28 DNA Genus/species-Inovirus M13MP18Descriptive title-Wild type M13MP18 region replacedATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACGGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTCCTATTGGTTAA SEQ ID NO: 29 DNAUnknown/artificial-commercially available from DNA2.0Descriptive title-Paprika sequenceATGGTGTCAAAGGGAGAAGAACTGATCAAAGAGAATATGAGGATGAAACTCTACATGGAAGGAACTGTGAACAACCACCATTTCAAGTGCACGAGCGAGGGTGAAGGGAAACCTTACGAAGGTACCCAGACCATGCGGATTAAGGTCGTCGAAGGAGGACCACTCCCCTTCGCATTCGACATCCTGGCCACTTCCTTCATGTACGGGTCGCGCACTTTCATCAAGTACCCAAAAGGGATCCCCGACTTCTTCAAGCAGTCCTTTCCGGAGGGATTCACTTGGGAACGCGTCACTAGATACGAGGATGGCGGAGTGGTCACCGTGATGCAAGACACCTCTTTGGAAGATGGATGCCTGGTGTACCACGTGCAAGTCAGAGGAGTGAACTTTCCGAGCAATGGGCCGGTGATGCAGAAGAAAACCAAGGGCTGGGAACCGAACACCGAAATGCTGTATCCAGCAGACGGAGGCTTGGAGGGCCGGTCCGACATGGCTCTGAAGCTTGTTGGAGGAGGACATCTGTCCTGCTCGTTCGTGACGACCTACCGGAGCAAGAAGCCGGCGAAAAACCTTAAGATGCCGGGGATCCACGCGGTGGATCATCGCCTGGAAAGGCTCGAGGAGTCAGACAACGAGATGTTTGTCGTGCAACGCGAGCACGCCGTGGCCCGCTACTGTGATCTCCCTTCAAAGCTGGGCCACAAGCTGAATTCCGGCCTCCGGTCGAGAGCCCAGGCTTCGAATTCAGCCGTGGACGGAACTGCGGGCCCTGGTTCGACCGGAAGCCGATGA SEQ ID NO: 30 DNAGenus/species-lambdalike lambdaDescriptive title-Wild type E. coli phage λ cII sequenceATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTGCTTAACAAAATCGCAATGCTTGGAACTGAGAAGACAGCGGAAGCTGTGGGCGTTGATAAGTCGCAGATCAGCAGGTGGAAGAGGGACTGGATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGACGACGACATGGCTCGATTGGCGCGACAAGTTGCTGCGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGASEQ ID NO: 31 Protein Synthetic (artificial/unknown)Descriptive title-NLS-FLAG-CAS9-His protein translated from SEQ ID NO: 27MPKKKRKVGDYKDDDDKLEPGEKPYKCPECGKSFSQSGALTRHQRTHTRDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNEVINFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHHSEQ ID NO: 32 DNA Genus/species-Cytomegalovirus HCMVDescriptive title-HCMV RL13 fragment post-editingATGGACTGGCGATTTACGGTTACGTGGACGATACTAATGTCCGCGTTGTCAGAAAGCTGCAATCAAACCTGTTCTTGTCAATGTCCCTGTAGTACTACCGTTAACTATTCAACTAGTACTGAGACAGCCACATCAACATACAGTACAACAGTTATCAGCAATAAAAGCACTTCAGAATCTATAAATTGCTCTACTGCAACTACACCAGCAAACACCGTTTCTACAAAACCGTCGGAAACAACCACACAGATATCCACAACGACGAACACAAACGTTGAGACTACCACATGTACCAACACCACCACGACCGTTACTTGTGATGGTTTCAATTATACAGTCCATAAAAGATGCGATCGCAGTTACGAGGTAATCAACGTAACAGGATACGTTGGTAGCAACATAACTCTAAAAAAATGCAATCAGACTGAGAAATGGCACAATGTAGACTGGATTCATTATGAGTACCCCACGCATAAAATGTGCGAATTAGGCAACTATCACCAAACCACACCACGGCACGACATATGTTTTGACTGCAACGACACCTCCCTAACTATCTACAACTTAACCACAAAAAACGCTGGAAAATATACCAGGCGTCACCGTGATAACGGTCAAGAAGAAAATTACTACGTAACGGTGTTAATTGGAGACACAACGTTATTCACTCTTGGCACATGCCCTGTAAGATATAAAGAATCTACGAACACTGAAAACACCATTGGAAGTAGCATCATAGAAACCATTGAGAAAGCTAACATTCCCCTGGGAATTCATGCTGTATGGGCAGGCGTAGTGGTATCAGTGGCGCTTATAGCGTTGTACATGGGTAGCCATCGCATTCCCAAAAAGCCGCATTACACCAAACTTCCCAAATATGATCCAGATGAATTTTGGACTAAGGCTTAA SEQ ID NO: 33 DNA Genus/species-Cytomegalovirus HCMVDescriptive title-HCMV RL13 fragment pre-editingATGGACTGGCGATTTACGGTTACGTGGACCGTTACTTGTGATGGTTTCAATTATACAGTCCATAAAAGATGCGATCGCAGTTACGAGGTAATCAACGTAACAGGATACGTTGGTAGCAACATAACTCTAAAAAAATGCAATCAGACTGAGAAATGGCACAATGTAGACTGGATTCATTATGAGTACCCCACGCATAAAATGTGCGAATTAGGCAACTATCACCAAACCACACCACGGCACGACATATGTTTTGACTGCAACGACACCTCCCTAACTATCTACAACTTAACCACAAAAAACGCTGGAAAATATACCAGGCGTCACCGTGATAACGGTCAAGAAGAAAATTACTACGTAACGGTGTTAATTGGAGACACAACGTTATTCACTCTTGGCACATGCCCTGTAAGATATAAAGAATCTACGAACACTGAAAACACCATTGGAAGTAGCATCATAGAAACCATTGAGAAAGCTAACATTCCCCTGGGAATTCATGCTGTATGGGCAGGCGTAGTGGTATCAGTGGCGCTTATAGCGTTGTACATGGGTAGCCATCGCATTCCCAAAAAGCCGCATTACACCAAACTTCCCAAATATGATCCAGATGAATTTTGGACTAAGGCTTAA SEQ ID NO: 34 ProteinGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 Gp13 protein sequenceMLALGAFDLSGLMVGSCLVVGGELKALCVDDRHSRQGIGAELVRAAELAGAEYLTCFEFLEPFYADLGWSTTHREANWTAGEPDVLHMRAPGHDV SEQ ID NO: 35 ProteinGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 Gp38 protein sequenceMARFKNPETIHVADGVEAVFSLDFPFLRREDVFVQVDKILVTDYTWVDDTNIQLAVVPKKDQEVRIFRDTPAQVPDTQFSQDIPFLPRYIDANNKQLLYAVQEGINTANLALDGVLDAIRIAEEARRLAQEALDAANEALRRALGFAEIRTVTEDSDIDPSWRGYWNRCITADKPLTLTMQMEDPDAPWVEFSEVHFEQAGVRDLNIVAGPGVTINRLQNTTMQLYGENGVCTLKRLGANHWIVFGAMEDE SEQ ID NO: 36 ProteinGenus/species-Phikmvlikevirus LUZ19Descriptive title-Wild type LUZ19 Gp40 protein sequenceMFKTEVKGRYTLIRRKADGTPVETLEFDNIITNAGLDWIAAMDTDLMGEPVAVSTSTADPNPSAPAIPEVVQRTSASAPGGGTTSGLDGEWLFWRRRWRFPQGTLAGQVLATVGLICNSDRRFESNTGELIPKDTPLSYTRIKDAAGQPTTLVVAADEILDVQYEFRSRPVGTAEAKFVISGVERTFRLIPKPFANRANLSGERYIFYNTNPYINGKDASGGNVRDGQWQKKYPKYVRGSYKAQITLLAQVQNGNMAGGITGTEELQIYNGRNYVLDINPPVVKNNTQEFTVTLEFTVARASEQ ID NO: 37 Protein Genus/species-Pseudomonas aeruginosaDescriptive title-PyoS5 protein sequenceMSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEAERKAAEEQALQDAIKFTADFYKEVTEKFGARTSEMARQLAEGARGKNIRSSAEAIKSFEKHKDALNKKLSLKDRQAIAKAFDSLDKQMMAKSLEKFSKGFGVVGKAIDAASLYQEFKISTETGDWKPFFVKIETLAAGAAASWLVGIAFATATATPIGILGFALVMAVTGAMIDEDLLEKANNLVISI SEQ ID NO: 38 ProteinGenus/species-Phikmvlikevirus LKD16Descriptive title-LKD16 Gp18 protein sequenceMRVPTEHERTLRCLLQD1HGPLNLLEPGIRVKVEEACLGYLGYRERGYWELRLQVDYDHPKLGHLRYSQAVPEYVLINDRDSIIKYLMEAVPRQVLEGMLNKAQEFVTKNWYSL SEQ ID NO: 39 ProteinGenus/species-Phikmvlikevirus LKA1Descriptive title-LKA1 Gp49 protein sequenceMAQTPSTWADYVGDGVEDTEQVTEPYQKQQEVEVTVGGDPAAFTEISAGWIQLAAVPVNGAAIRVRRSTEAFEPRHEEANGVPLLPRFIDENNTQFLYTVQEAVNETHGIASEALSVAEEARGIAQAASDKVDAATIDSAHQLRLDLADPAKGPGLLGYDRDVSYPVGSVGQSLQFLEMGRVTPAQFGAVGDGASHPLSERYATLAEAQTVYPHAVALSDEIDWAALQAAVDSGAPVHIPSGDYQINRGISSTGSLQIAGDGATSIIRPTAAFTGTSVLSCVGSLVALPNISSVSAGSLTIDEASTPNLVAGDVEIIYNPTDSSFSGFRTSYRAGEFCEVRAVSGNTVTIRSALYAAYDGATVAIYKVVSGVVDIASIQIVGGTVPMNGLLVEAVVSPRVDDVTVTLANNAGVYEARCYDAKITNSNISNIGDGGDDYGIIEGNCHDGGADNCKVYARRHAIATGGDAEVGCVPVRNVRMRNCTLRNDITSGTHCADFHGNAEDCSYENCTIYGGATWQGKDISYRHCTITNASGGWIVISAEILGGTFLLDQCTLYTTGDPQPGNRGVIDVGGNSAVLTTNTTQPCNFLIQGGSLRAPSLSTSSYLLRARLEGSTVPVNIQYSGQAIDVGSLGKVLQLDITSGSTSPEYLIVENLAGLPSGITLASAAGGFASAPMRMPVLGGRVQVTTATNASSVTAPVTFRYIYPKAPTVQVTKTDRSYAGNRVGVAIANPTSASGATLGLFTDDGTNFSSAVTNQLNWQAGIYEV SEQ ID NO: 40Protein Genus/species-Phikmvlikevirus NTUH-K2044-K1-1Descriptive title-NTUH-K2044-K1-1 Gp34 protein sequenceMALIRLVAPERVFSDLASMVAYPNFQVQDKITLLGSAGGDETETTTASVVDNGTVEAVPGGYLLRKEVGPAYSSWFSNWTGIVTFMSAPNRHLVVDTVLQATSVLNIKSNSTLEFTDTGRILPDAAVARQVLNITGSAPSVFVPLAADAAAGSKVITVAAGALSAVKGTYLYLRSNKLCDGGPNTYGVKISQIRKVVGVSTSGGVTSIRLDKALHYNYYLSDAAEVGIPTMVENVTLVSPYINEEGYDDLNREFTSGISANFAADLHIQDGVIIGNKRPGASDIEGRSAIKENNCVDSTVKGTCFYNIGWYGVEVLGCSEDTEVHDIHAMDVRHAISLNWQSTADGDKWGEPIEFLGVNCEAYSTTQAGEDTHDIGKRVKEVRCVSYDSADDGEQARTNGVEYLNCRAYRAAMDGEASNTGVAEPIYRECLAYDNVRSGENCSYGGGYVYDCEAHGSQNGVRINGGRVKGGRYTRNSSSHIFVTKDVAETAQTSLEEDGVSMRYDGTGRAVYFHGTVGIDPTLVSMSNNDMTGHGLFWALLSGYTVQPTPPRMSRNLLDDTGIRGVATLVAGEATVNARVRGNEGSVANSFKWVSEVKLTRLTFPSSAGALTVTSVAQNQDVPTPNPDLNSFVIRSSNAADVSQVAWEVYL SEQ ID NO: 41 ProteinGenus/species-T7-like Pp15 Descriptive title-Pp15 Gp44 protein sequenceMARTIVQNALTGGQQDFEVPFDYILQRFVKLTLIGDGNRQELVLGTDERFIGPRTVRTNVEWGPAQGYTSIEIRRVTSASDRRVEFSDGSILTAGDLNIAQLQAIHIAEEARDSATENLSPDADGNYDARGARIYNLGDAVQPKDAVNRYTLDLAIAAALAMNTGNPNNAQNISYTPNGPGQSIRSVEGRLRDAVFVSDYMTTPRDGVTSNQQDLEKALAAANAKGADLFWPDDIPFESTSPLALIHAVYHVGRGVINANGTLFYVNPKNGQHNRLHVSPGGTGDGLAAGRPLGTIWSALAALNMRAPLTTRWSLEMTAGAYNEAVTLPNYLTSCNDYLAFNWPNTGQERMEPTAYPSALDGTGQTGLTGFHTGIGNRITINNVCMSNWYDTALTPTQQVRRAFVVGAYSTAYVVNCAFIYNGIASVSVLPGGTAIVTGGIVDGGRFGLDNTGGRLSLTATKSNYTQVRNCLEYGLYSKHDASTVMDNTEERNCGNHPAAVAYGAAIFAYKENCSVDTRGVKFYGNNIAQHCRGGITSDNPGDPDIYGTGADANKRLFLCTGGGSDDIQFYEARRVMDITKRTGGGSTTASVSSLLLAAVASVRKGYFAHNDQVIRMTLMFRATGSAGIFTPTLRTPLGTIPLGSFRVASGQYGEEKLTIRPTLTSDGLIVGFSCINAVQNLGSSVGQIIVSGTVDLRTVDQLVEMWGYSEAGGTASYIQGLIELVG SEQ ID NO: 42 ProteinGenus/species-Aggregatibacter actinomycetemcomitansDescriptive title-DspB protein sequenceMNCCVKGNSIYPQKTSTKQTGLMLDIARHEYSPEVIKSFIDTISLSGGNELHLHESDHENYAIESHLLNQRAENAVQGKDGIYINPYTGKPFLSYRQLDDIKAYAKAKGIELIPELDSPNHMTAIFKLVQKDRGVKYLQGLKSRQVDDEIDITNADSITEMQSLMSEVIDIFGDTSQHFHIGGDEFGYSVESNHEFITYANKLSYFLEKKGLKTRMWNDGLIKNTFEQINPNIEITYWSYDGDTQDKNEAAERRDMRVSLPELLAKGETVLNYNSYYLYIVPKASPTESQDAAFAAKDVIKNWDLGVWDGRNTKNRVQNTHEIAGAALSIWGEDAKALKDETIQKNTKSLLEAVIIIKTNGDE SEQ ID NO: 43 ProteinGenus/species-Staphylococcus aureusDescriptive title-SaPSMa3 protein sequence MEFVAKLFKFFKDLLGKFLGNNSEQ ID NO: 44 Protein Genus/species-Staphylococcus aureusDescriptive title-SaPAMb2 protein sequenceMTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGLLGKLFGF SEQ ID NO: 45 ProteinGenus/species-Staphylococcus epidermidisDescriptive title-SePSMa protein sequence MADVIAKIVEIVKGLIDQFTQKSEQ ID NO: 46 Protein Genus/species-Levivirus M52Descriptive title-MS2 L protein sequenceMETRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLTSEQ ID NO: 47 Protein Genus/species-Levivirus PRR1Descriptive title-PRR1 L protein sequenceMCKVSTKVDSKLTESVGQLTIRSYLWLRNILALAGLLFVILLATNHL SIAIYSP SEQ ID NO: 48Protein Genus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 Gp18 protein sequenceMRMPTEEERMIRCLLADIHEPLDLLFPGLRTKAHMDPQAEELSIRIDYDHAKLGRMGFCHAVSLYQLSIYGREGMVRYLMQEIPRRVLEGLLVKAQQYSQSNWYSK SEQ ID NO: 49 ProteinGenus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 Gp49 protein sequenceMSKAKLRVIADTPELESVLKALLTATYAIEDLLNEAVASKVLNSRLGWSAVGEYVELFNRTQSRVAGLIPESEQ ID NO: 50 DNA Genus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 gp18 gene sequenceATGAGAATGCCAACCGAAGAAGAACGCATGATCCGCTGTTTACTGGCGGATATCCACGAGCCACTGGACCTGCTGTTCCCCGGCCTCCGTACCAAGGCCCATATGGACCCGCAAGCAGAGGAACTGTCGATTCGAATTGACTACGACCATGCGAAGCTGGGCCGTATGGGATTCTGCCACGCGGTATCCCTATATCAACTGTCCATATATGGCCGCGAGGGGATGGTCCGCTACCTGATGCAGGAGATTCCCCGCCGCGTGCTGGAAGGTCTGCTGGTCAAGGCGCAGCAGTACAGCCAAAGCAACTGGTACAGCAAATGA SEQ ID NO: 51 DNAGenus/species-Phikmvlikevirus LUZ19Descriptive title-LUZ19 Gp49 protein sequenceATGAGCAAAGCCAAACTACGAGTCATCGCCGACACCCCGGAGCTGGAGTCAGTGCTAAAAGCATTGCTGACCGCCACCTACGCTATCGAGGACCTGCTCAACGAGGCCGTGGCTAGCAAGGTGCTAAACTCCCGCCTGGGCTGGTCCGCAGTCGGCGAGTATGTCGAACTGTTCAACCGCACGCAATCCCGCGTGGCCGGGTTGATTCCCGAGTAG

What is claimed is:
 1. A method for generating a recombinant phage thatexpresses two or more payloads, the method comprising: (a) providing afirst viral genome from a first phage; and (b) generating an engineeredviral genome by (1) in vitro digesting a region of the first viralgenome using an endonuclease; and (2) assembling at least one fragmentof the digested first viral genome with at least one repair nucleic acidmolecule, to generate a second viral genome comprising at least onemodification compared to the first and the second viral genome, uponintroduction into a host cell, produces viral particles with two or morepayloads selected from the group consisting of a DNase, anexopolysaccharide (EPS) depolymerase, and one or more surfactant phenolsoluble modulin (PSM); and (c) introducing said second viral genome intoa host cell capable of producing viral particles, thereby producing anengineered recombinant phage.
 2. The method of claim 1, furthercomprising: (3) repeating steps (a)-(b) in one or more iterations. 3.The method of claim 1, wherein each payload independently changes one ormore of the host range, viral lytic cycle, adsorption, attachment,injection, replication and assembly, lysis, burst size, immune evasion,immune stimulation, immune deactivation, biofilm dispersion, bacterialphage resistance, bacterial antibiotic sensitization, modulation ofvirulence factors, and targeted host genome digestion or editing of therecombinant phage as compared to the first phage.
 4. The method of claim1, wherein the first viral genome is isolated from viral particles. 5.The method of claim 1, wherein the first viral genome or the at leastone repair nucleic acid molecule is synthesized de novo.
 6. The methodof claim 5, wherein de novo synthesis comprises combining chemicallysynthesized nucleic acid molecules, PCR-amplified nucleic acidsequences, digested fragments of isolated nucleic acid molecules, or anycombination thereof.
 7. The method of claim 5, wherein the first viralgenome or the at least one repair nucleic acid molecule is amplifiedprior to in vitro digestion.
 8. The method of claim 1, wherein the firstviral genome is at least 3 kb, at least 10 kb, at least 18 kb, at least25 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 70 kb,at least 100 kb, at least 125 kb, at least 150 kb, at least 200 kb, orat least 300 kb.
 9. The method of claim 1, wherein the assembly isperformed in vitro or in vivo.
 10. The method of claim 9, wherein theassembly is performed in vitro with a mixture comprising: (a) anisolated 5′ to 3′ exonuclease that lacks 3′ exonuclease activity; (b) anisolated non-strand-displacing DNA polymerase with 3′ exonucleaseactivity, or a mixture of said DNA polymerase with a second DNApolymerase that lacks 3′ exonuclease activity; (c) an isolated ligase;and (d) a mixture of dNTPs, under conditions that are effective forinsertion of the fragment into the digested viral nucleic acid to form arecombinant nucleic acid comprising the engineered viral genome.
 11. Themethod of claim 1, wherein the endonuclease is an RNA-guided nuclease.12. The method of claim 11, further comprising at least one guiding RNA.13. The method of claim 12, wherein the RNA-guided nuclease is Cas9 or aCas9 derived enzyme, and wherein the at least one guiding RNAcomprises 1) a chimeric gRNA or 2) a crRNA and tracrRNA.
 14. The methodof claim 1, wherein the endonuclease is heat inactivated or removedprior to assembly.
 15. The method of claim 1, wherein the in vitrodigestion further comprises spermidine.
 16. The method of claim 1,wherein introducing comprises transforming the engineered viral genomeinto a host cell.
 17. The method of claim 1, further comprising using anin vitro packaging kit for packaging of the engineered viral genome intoviral particles.
 18. A method of engineering a recombinant nucleic acidsequence comprising: (a) providing a nucleic acid; (b) in vitrodigesting a region of the nucleic acid using an RNA-guided nuclease; and(c) assembling a recombinant nucleic acid by insertion of a DNA fragmentinto the digested nucleic acid, wherein assembling is performed in vitroin a single vessel with a mixture of components comprising: (i) anisolated 5′ to 3′ exonuclease that lacks 3′ exonuclease activity; (ii)an isolated non-strand-displacing DNA polymerase with 3′ exonucleaseactivity, or a mixture of said DNA polymerase with a second DNApolymerase that lacks 3′ exonuclease activity; (iii) an isolated ligase;and (iv) a mixture of dNTPs; under conditions that are effective forinsertion of the fragment into the digested nucleic acid to form arecombinant nucleic acid sequence.
 19. The method of claim 18, whereinthe RNA-guided nuclease is Cas9 or a Cas9 derived enzyme.
 20. The methodof claim 18, wherein the RNA-guided nuclease is heat inactivated orremoved prior to assembly.
 21. The method of claim 18, furthercomprising: (d) transforming the recombinant nucleic acid into a hostcell.
 22. The method of claim 18, wherein the nucleic acid is a plasmidisolated from a host cell.
 23. The method of claim 22, wherein theplasmid is at least 6 kb.
 24. The method of claim 22, wherein theplasmid is at least 10 kb.
 25. The method of claim 22, wherein theplasmid is at least 15 kb.
 26. The method of claim 22, wherein theplasmid is at least 20 kb.
 27. The method of claim 1, wherein the two ormore payloads comprise a DNase.
 28. The method of claim 1, wherein saidtwo or more payloads comprise an EPS depolymerase.
 29. The method ofclaim 1, wherein said two or more payloads comprise a phenol solublemodulin.
 30. The method of claim 29, wherein said phenol soluble modulinis selected from the group consisting of PSMα, PSMα3, and PSMβ32.